Enzymatic DNA molecules

ABSTRACT

The present invention discloses deoxyribonucleic acid enzymes—catalytic or enzymatic DNA molecules—capable of cleaving nucleic acid sequences or molecules, particularly RNA, in a site-specific manner, as well as compositions including same. Methods of making and using the disclosed enzymes and compositions are also disclosed.

TECHNICAL FIELD

The present invention relates to nucleic acid enzymes or catalytic(enzymatic) DNA molecules that are capable of cleaving other nucleicacid molecules, particularly RNA. The present invention also relates tocompositions containing the disclosed enzymatic DNA molecules and tomethods of making and using such enzymes and compositions.

BACKGROUND

The need for catalysts that operate outside of their native context orwhich catalyze reactions that are not represented in nature has resultedin the development of “enzyme engineering” technology. The usual routetaken in enzyme engineering has been a “rational design” approach,relying upon the understanding of natural enzymes to aid in theconstruction of new enzymes. Unfortunately, the state of proficiency inthe areas of protein structure and chemistry is insufficient to make thegeneration of novel biological catalysts routine.

Recently, a different approach for developing novel catalysts has beenapplied. This method involves the construction of a heterogeneous poolof macromolecules and the application of an in vitro selection procedureto isolate molecules from the pool that catalyze the desired reaction.Selecting catalysts from a pool of macromolecules is not dependent on acomprehensive understanding of their structural and chemical properties.Accordingly, this process has been dubbed “irrational design” (Brennerand Lerner, PNAS USA 89: 5381-5383 (1992)).

Most efforts to date involving the rational design of enzymatic RNAmolecules or ribozymes have not led to molecules with fundamentally newor improved catalytic function. However, the application of irrationaldesign methods via a process we have described as “directed molecularevolution” or “in vitro evolution”, which is patterned after Darwinianevolution of organisms in nature, has the potential to lead to theproduction of DNA molecules that have desirable functionalcharacteristics.

This technique has been applied with varying degrees of success to RNAmolecules in solution (see, e.g., Mills, et al., PNAS USA 58: 217(1967); Green, et al., Nature 347: 406 (1990); Chowrira, et al., Nature354: 320 (1991); Joyce, Gene 82: 83 (1989); Beaudry and Joyce, Science257: 635-641 (1992); Robertson and Joyce, Nature 344: 467 (1990)), aswell as to RNAs bound to a ligand that is attached to a solid support(Tuerk, et al., Science 249: 505 (1990); Ellington, et al., Nature 346:818 (1990)). It has also been applied to peptides attached directly to asolid support (Lam, et al., Nature 354: 82 (1991)); and to peptideepitopes expressed within a viral coat protein (Scott, et al., Science249: 386 (1990); Devlin, et al., Science 249: 249 (1990); Cwirla, etal., PNAS USA 87: 6378 (1990)).

It has been more than a decade since the discovery of catalytic RNA(Kruger, et al., Cell 31: 147-157 (198:7); Guerrier-Takada, et al., Cell35: 849-857 (1983)). The list of known naturally-occurring ribozymescontinues to grow (see Cech, in The RNA World, Gesteland & Atkins(eds.), pp. 239-269, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1993); Pyle, Science 261: 709-714 (1993); Symons, Curr.Opin. Struct. Biol. 4: 322-330 (1994)) and, in recent years, has beenaugmented by synthetic ribozymes obtained through in vitro evolution.(See, e.g., Joyce, Curr. Opin. Struct. Biol. 4: 331-336 (1994); Breaker& Joyce, Trends Biotech. 12: 268-275 (1994); Chapman & Szostak, Curr.Opin. Struct. Biol. 4: 618-622 (1994).)

It seems reasonable to assume that DNA can have catalytic activity aswell, considering that it contains most of the same functional groups asRNA. However, with the exception of certain viral genomes andreplication intermediates, nearly all of the DNA in biological organismsoccurs as a complete duplex, precluding it from adopting a complexsecondary and tertiary structure. Thus it is not surprising that DNAenzymes have not been found in nature.

Until the advent of the present invention, the design, synthesis and useof catalytic DNA molecules with nucleotide-cleaving capabilities has notbeen disclosed or demonstrated. Therefore, the discoveries andinventions disclosed herein are particularly significant, in that theyhighlight the potential of in vitro evolution as a means of designingincreasingly more efficient catalytic molecules, including enzymatic DNAmolecules that cleave other nucleic acids, particularly RNA.

BRIEF SUMMARY OF THE INVENTION

The present invention thus contemplates a synthetic or engineered (i.e.,non-naturally-occurring) catalytic DNA molecule (or enzymatic DNAmolecule) capable of cleaving a substrate nucleic acid (NA) sequence ata defined cleavage site. The invention also contemplates an enzymaticDNA molecule having an endonuclease activity.

In one preferred variation, the endonuclease activity is specific for anucleotide sequence defining a cleavage site comprising single-strandednucleic acid in a substrate nucleic acid sequence. In another preferredvariation, the cleavage site is double-stranded nucleic acid. Similarly,substrate nucleic acid sequences may be single-stranded,double-stranded, partially single- or double-stranded, looped, or anycombination thereof.

In another contemplated embodiment, the substrate nucleic acid sequenceincludes one or more nucleotide analogues. In one variation, thesubstrate nucleic acid sequence is a portion of, or attached to, alarger molecule.

In various embodiments, the larger molecule is selected from the groupconsisting of RNA, modified RNA, DNA, modified DNA, nucleotide analogs,or composites thereof. In another example, the larger molecule comprisesa composite of a nucleic acid sequence and a non-nucleic acid sequence.

In another embodiment, the invention contemplates that a substratenucleic acid sequence includes one or more nucleotide analogs. A furthervariation contemplates that the single stranded nucleic acid comprisesRNA, DNA, modified RNA, modified DNA, one or more nucleotide analogs, orany composite thereof. In one embodiment of the disclosed invention, theendonuclease activity comprises hydrolytic cleavage of a phosphoesterbond at the cleavage site.

In various preferred embodiments, the catalytic DNA molecules of thepresent invention are single-stranded in whole or in part. Thesecatalytic DNA molecules may preferably assume a variety of shapesconsistent with their catalytic activity. Thus, in one variation, acatalytic DNA molecule of the present invention includes one or morehairpin loop structures. In yet another variation, a catalytic DNAmolecule may assume a shape similar to that of “hammerhead” ribozymes.In still other embodiments, a catalytic DNA molecule may assume aconformation similar to that of Tetrahymena thermophila ribozymes, e.g.,those derived from group I introns.

Similarly, preferred catalytic DNA molecules of the present inventionare able to demonstrate site-specific endonuclease activity irrespectiveof the original orientation of the substrate molecule. Thus, in onepreferred embodiment, an enzymatic DNA molecule of the present inventionis able to cleave a substrate nucleic acid sequence that is separatefrom the enzymatic DNA molecule—i.e., it is not linked to the DNAzyme.In another preferred embodiment, an enzymatic DNA molecule is able tocleave an attached substrate nucleic acid sequence—i.e., it is able toperform a reaction similar to self-cleavage.

The invention also contemplates enzymatic DNA molecules (catalytic DNAmolecules, deoxyribozymes or DNAzymes) having endonuclease activity,whereby the endonuclease activity requires the presence of a divalentcation. In various preferred, alternative embodiments, the divalentcation is selected from the group consisting of Pb²⁺, Mg²⁺, Mn²⁺, Zn²⁺,and Ca²⁺. Another variation contemplates that the endonuclease activityrequires the presence of a monovalent cation. In such alternativeembodiments, the monovalent cation is preferably selected from the groupconsisting of Na⁺ and K⁺.

In various preferred embodiments of the invention, an enzymatic DNAmolecule comprises a nucleotide sequence selected from the groupconsisting of SEQ ID NO 3, SEQ ID NO 14; SEQ ID NO 15; SEQ ID NO 16; SEQID NO 17; SEQ ID NO 18; SEQ ID NO 19; SEQ ID NO 20; SEQ ID NO 21; andSEQ ID NO 22. In other preferred embodiments, a catalytic DNA moleculeof the present invention comprises a nucleotide sequence selected fromthe group consisting of SEQ ID NO 23; SEQ ID NO 24; SEQ ID NO 25; SEQ IDNO 26; SEQ ID NO 27; SEQ ID NO 28; SEQ ID NO 29; SEQ ID NO 30; SEQ ID NO31; SEQ ID NO 32; SEQ ID NO 33; SEQ ID NO 34; SEQ ID NO 35; SEQ ID NO36; SEQ ID NO 37; SEQ ID NO 38; and SEQ ID NO 39.

Another preferred embodiment contemplates that a catalytic DNA moleculeof the present invention comprises a nucleotide sequence selected fromthe group consisting of SEQ ID NO 50 and SEQ ID NO 51. In yet anotherpreferred embodiment, a catalytic DNA molecule of the present inventioncomprises a nucleotide sequence selected from the group consisting ofSEQ ID NOS 52 through 101. As disclosed herein, catalytic DNA moleculeshaving sequences substantially similar to those disclosed herein arealso contemplated. Thus, a wide variety of substitutions, deletions,insertions, duplications and other mutations may be made to thewithin-described molecules in order to generate a variety of otheruseful enzymatic DNA molecules; as long as said molecules displaysite-specific cleavage activity as disclosed herein, they are within theboundaries of this disclosure.

In a further variation of the present invention, an enzymatic DNAmolecule of the present invention preferably has a substrate bindingaffinity of about 1 μM or less. In another embodiment, an enzymatic DNAmolecule of the present invention binds substrate with a K_(D) of lessthan about 0.1 μM.

The present invention also discloses enzymatic DNA molecules havinguseful turnover rates. In one embodiment, the turnover rate is less than5 hr⁻¹; in a preferred embodiment, the rate is less than about 2 hr⁻¹;in a more preferred embodiment, the rate is less than about 1 hr⁻¹: inan even more preferred embodiment, the turnover rate is about 0.6 hr⁻¹or less.

In still another embodiment, an enzymatic DNA molecule of the presentinvention displays a useful turnover rate wherein the k_(obs) is lessthan 1 min⁻¹, preferably less than 0.1 min⁻¹; more preferably, less than0.01 min⁻¹; and even more preferably, less than 0.005 min⁻¹. In onevariation, the value of k_(obs) is approximately 0.002 min⁻¹ or less.

The present invention also contemplates embodiments in which thecatalytic rate of the disclosed DNA enzymes is fully optimized. Thus, invarious preferred embodiments, the K_(m) for reactions enhanced by thepresence of Mg²⁺ is approximately 0.5-20 mM, preferably about 1-10 mM,and more preferably about 2-5 mM.

The present invention also contemplates an embodiment whereby thenucleotide sequence defining the cleavage site comprises at least onenucleotide. In various other preferred embodiments, a catalytic DNAmolecule of the present invention is able to recognize and cleave anucleotide sequence defining a cleavage site of two or more nucleotides.

In various preferred embodiments, an enzymatic DNA molecule of thepresent invention comprises a conserved core flanked by one or moresubstrate binding regions. In one embodiment, an enzymatic DNA moleculeincludes first and second substrate binding regions. In anotherembodiment, an enzymatic DNA molecule includes two or more substratebinding regions.

As noted previously, preferred catalytic DNA molecules of the presentinvention may also include a conserved core. In one preferredembodiment, the conserved core comprises one or more conserved regions.In other preferred variations, the one or more conserved regions includea nucleotide sequence selected from the group consisting of CG; CGA;AGCG; AGCCG; CAGCGAT; CTTGTTT; and CTTATTT (see, e.g., FIG. 3).

In one embodiment of the invention, an enzymatic DNA molecule of thepresent invention further comprises one or more variable or spacernucleotides between the conserved regions in the conserved core. Inanother embodiment, an enzymatic DNA molecule of the present inventionfurther comprises one or more variable or spacer nucleotides between theconserved core and the substrate binding region.

In one variation, the first substrate binding region preferably includesa nucleotide sequence selected from the group consisting of CATCTCT;GCTCT; TTGCTTTTT; TGTCTTCTC; TTGCTGCT; GCCATGCTTT (SEQ ID NO 40);CTCTATTTCT (SEQ ID NO 41); GTCGC3CA; CATCTCTTC; and ACTTCT. In anotherpreferred variation, the second substrate binding region includes anucleotide sequence selected from the group consisting of TATGTGACGCTA(SEQ ID NO 42); TATAGTCGTA (SEQ ID NO 43); ATAGCGTATTA (SEQ ID NO 44);ATAGTTACGTCAT (SEQ ID NO 45); AATAGTGAAGTGTT (SEQ ID NO 46); TATAGTGTA;ATAGTCGGT; ATAGGCCCGGT (SEQ ID NO 47); AATAGTGAGGCTTG (SEQ ID NO 48);and ATGNTG.

In various embodiments of the present invention, the substrate bindingregions vary in length. Thus, for example, a substrate binding regionmay comprise a single nucleotide to dozens of nucleotides. However, itis understood that substrate binding regions of about 3-25 nucleotidesin length, preferably about 3-15 nucleotides in length, and morepreferably about 3-10 nucleotides in length are particularly preferred.In various embodiments, the individual nucleotides in the substratebinding regions are able to form complementary base pairs with thenucleotides of the substrate molecules; in other embodiments,noncomplementary base pairs are formed. A mixture of complementary andnoncomplementary base pairing is also contemplated as falling within thescope of the disclosed embodiments of the invention.

In another preferred embodiment, a catalytic DNA molecule of the presentinvention may further comprise a third substrate binding region. In somepreferred embodiments, the third region includes a nucleotide sequenceselected from the group consisting of TGTT; TGTTA; and TGTTAG. Anotherpreferred embodiment of the present invention discloses an enzymatic DNAmolecule further comprising one or more variable or “spacer” regionsbetween the substrate binding regions.

In another disclosed embodiment, the present invention contemplates apurified, synthetic enzymatic DNA molecule separated from other DNAmolecules and oligonucleotides, the enzymatic DNA molecule having anendonuclease activity, wherein the endonuclease activity is specific fora nucleotide sequence defining a cleavage site comprising single- ordouble-stranded nucleic acid in a substrate nucleic acid sequence. Inone variation, a synthetic (or engineered) enzymatic DNA molecule havingan endonuclease activity is disclosed, wherein the endonuclease activityis specific for a nucleotide sequence defining a cleavage siteconsisting essentially of a single- or double-stranded region of asubstrate nucleic acid sequence.

In yet another embodiment, the invention contemplates an enzymatic DNAmolecule comprising a deoxyribonucleotide polymer having a catalyticactivity for hydrolyzing a nucleic acid-containing substrate to producesubstrate cleavage products. In one variation, the hydrolysis takesplace in a site-specific manner. As noted previously, the polymer may besingle-stranded, double-stranded, or some combination of both.

The invention further contemplates that the substrate comprises anucleic acid sequence. In various embodiments, the nucleic acid sequencesubstrate comprises RNA, modified RNA, DNA, modified DNA, one or morenucleotide analogs, or composites of any of the foregoing. Oneembodiment contemplates that the substrate includes a single-strandedsegment; still another embodiment contemplates that the substrate isdouble-stranded.

The present invention also contemplates an enzymatic DNA moleculecomprising a deoxyribonucleotide polymer having a catalytic activity forhydrolyzing a nucleic acid-containing substrate to produce a cleavageproduct. In one variation, the enzymatic DNA molecule has an effectivebinding affinity for the substrate and lacks an effective bindingaffinity for the cleavage product.

In one preferred embodiment, the invention discloses anon-naturally-occurring enzymatic DNA molecule comprising a nucleotidesequence defining a conserved core flanked by recognition domains,variable regions, and spacer regions. Thus, in one preferred embodimentthe nucleotide sequence defines a first variable region contiguous oradjacent to the 5′-terminus of the molecule, a first recognition domainlocated 3′-terminal to the first variable region, a first spacer regionlocated 3′-terminal to the first recognition domain, a first conservedregion located 3′-terminal to the first spacer region, a second spacerregion located 3′-terminal to the first conserved region, a secondconserved region located 3′-terminal to the second spacer region, asecond recognition domain located 3′-terminal to the second conservedregion, and a second variable region located 3′-terminal to the secondrecognition domain.

In another embodiment, the nucleotide sequence preferably defines afirst variable region contiguous or adjacent to the 5′-terminus of themolecule, a first recognition domain located 3′-terminal to the firstvariable region, a first spacer region located 3′-terminal to the firstrecognition domain, a first conserved region located 3′-terminal to thefirst spacer region, a second spacer region located 3′-terminal to thefirst conserved region, a second conserved region located 3′-terminal tothe second spacer region, a second recognition domain located3′-terminal to the second conserved region, a second variable regionlocated 3′-terminal to the second recognition domain, and a thirdrecognition domain located 3′-terminal to the second variable region.

In one variation of the foregoing, the molecule includes a conservedcore region flanked by two substrate binding domains; in another, theconserved core region comprises one or more conserved domains. In otherpreferred embodiments, the conserved core region further comprises oneor more variable or spacer nucleotides. In yet another embodiment, anenzymatic DNA molecule of the present invention further comprises one ormore spacer regions.

The present invention further contemplates a wide variety ofcompositions. For example, compositions including an enzymatic DNAmolecule as described hereinabove are disclosed and contemplated herein.In one alternative embodiment, a composition according to the presentinvention comprises two or more populations of enzymatic DNA moleculesas described above, wherein each population of enzymatic DNA moleculesis capable of cleaving a different sequence in a substrate. In anothervariation, a composition comprises two or more populations of enzymaticDNA molecules as described hereinabove, wherein each population ofenzymatic DNA molecules is capable of recognizing a different substrate.In various embodiments, it is also preferred that compositions include amonovalent or divalent cation.

The present invention further contemplates methods of generating,selecting, and isolating enzymatic DNA molecules of the presentinvention. In one variation, a method of selecting enzymatic DNAmolecules that cleave a nucleic acid sequence (e.g., RNA) at a specificsite comprises the following steps: (a) obtaining a population ofputative enzymatic DNA molecules—whether the sequences arenaturally-occurring or synthetic—and preferably, they aresingle-stranded DNA molecules; (b) admixing nucleotide-containingsubstrate sequences with the aforementioned population of DNA moleculesto form an admixture; (c) maintaining the admixture for a sufficientperiod of time and under predetermined reaction conditions to allow theputative enzymatic DNA molecules in the population to cause cleavage ofthe substrate sequences, thereby producing substrate cleavage products;(d) separating the population of DNA molecules from the substratesequences and substrate cleavage products; and (e) isolating DNAmolecules that cleave substrate nucleic acid sequences (e.g., RNA) at aspecific site from the population.

In a further variation of the foregoing method, the DNA molecules thatcleave substrate nucleic acid sequences at a specific site are taggedwith an immobilizing agent. In one example, the agent comprises biotin.

In yet another variation of the aforementioned method, one begins byselecting a sequence—e.g., a predetermined “target” nucleotidesequence—that one wishes to cleave using an enzymatic DNA moleculeengineered for that purpose. Thus, in one embodiment, the pre-selected(or predetermined) “target” sequence is used to generate a population ofDNA molecules capable of cleaving substrate nucleic acid sequences at aspecific site via attaching or “tagging” it to a deoxyribonucleic acidsequence containing one or more randomized sequences or segments. In onevariation, the randomized sequence is about 40 nucleotides in length; inanother variation, the randomized sequence is about 50 nucleotides inlength. Randomized sequences that are 1-40, 40-50, and 50-100nucleotides in length are also contemplated by the present invention.

In one embodiment of the present invention, the nucleotide sequence usedto generate a population of enzymatic DNA molecules is selected from thegroup consisting of SEQ ID NO 4, 23, 50 AND 51. In another embodiment,the “target” or “substrate” nucleotide sequence comprises a sequence ofone or more ribonucleotides—see, e.g., the relevant portions of SEQ IDNOS 4 and 23, and SEQ ID NO 49. It is also contemplated by the presentinvention that a useful “target” or “substrate” nucleotide sequence maycomprise DNA, RNA, or a composite thereof.

The invention also contemplates methods as described above, wherein theisolating step further comprises exposing the tagged DNA molecules to asolid surface having avidin linked thereto, whereby the tagged DNAmolecules become attached to the solid surface. As before, the substratemay be RNA, DNA, a composite of both, or a molecule including nucleotidesequences.

The present invention also contemplates a method for specificallycleaving a substrate nucleic acid sequence at a particular cleavagesite, comprising the steps of (a) providing an enzymatic DNA moleculecapable of cleaving a substrate nucleic acid sequence at a specificcleavage site; and (b) contacting the enzymatic DNA molecule with thesubstrate nucleic acid sequence to cause specific cleavage of thenucleic acid sequence at the cleavage site. In one variation, theenzymatic DNA molecule is a non-naturally-occurring (or synthetic) DNAmolecule. In another variation, the enzymatic DNA molecule issingle-stranded.

In still another variation of the foregoing method, the substratecomprises a nucleic acid. In various embodiments, the substrate nucleicacid comprises RNA, modified RNA, DNA, modified DNA, one or morenucleotide analogs, or composites of any of the foregoing. In yetanother embodiment, the specific cleavage is caused by the endonucleaseactivity of the enzymatic DNA molecule. Alteration of reactionconditions—e.g., the adjustment of pH, temperature, percent cation,percent enzyme, percent substrate, and percent product—is alsocontemplated herein.

The present invention also contemplates a method of cleaving aphosphoester bond, comprising (a) admixing an catalytic DNA moleculecapable of cleaving a substrate nucleic acid sequence at a definedcleavage site with a phosphoester bond-containing substrate, to form areaction admixture; and (b) maintaining the admixture underpredetermined reaction conditions to allow the enzymatic DNA molecule tocleave the phosphoester bond, thereby producing a population ofsubstrate products. In one embodiment, the enzymatic DNA molecule isable to cleave the phosphoester bond in a site-specific manner. Inanother embodiment, the method further comprises the steps of (c)separating the products from the catalytic DNA molecule; and (d) addingadditional substrate to the enzymatic DNA molecule to form a newreaction admixture.

The present invention also contemplates methods of engineering enzymaticDNA molecules that cleave phosphoester bonds. One exemplary methodcomprises the following steps: (a) obtaining a population ofsingle-stranded DNA molecules; (b) introducing genetic variation intothe population to produce a variant population; (c) selectingindividuals from the variant population that meet predeterminedselection criteria; (d) separating the selected individuals from theremainder of the variant population; and (e) amplifying the selectedindividuals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a selective amplification scheme for isolation ofDNAs that cleave a target RNA phosphoester. As shown, double-strandedDNA that contains a stretch of 50 random nucleotides (the molecule with“N₅₀” indicated above it) is amplified by PCR, employing a5′-biotinylated DNA primer that is terminated at the 3′ end by anadenosine ribonucleotide (rA). (The biotin label is indicated via theencircled letter “B”.) This primer is extended by Taq polymerase toyield a DNA product that contains a single embedded ribonucleotide. Theresulting double-stranded DNA is immobilized on a streptavidin matrixand the unbiotinylated DNA strand is removed by washing with 0.2 N NaOH.After re-equilibrating the column with a buffered solution, the columnis washed with the same solution with added 1 mM PbOAc. DNAs thatundergo Pb²⁺-dependent self-cleavage are released from the column,collected in the eluant, and amplified by PCR. The PCR products are thenused to initiate the next round of selective amplification.

FIG. 2 illustrates self-cleavage activity of the starting pool of DNA(G0) and populations obtained after the first through fifth rounds ofselection (G1-G5), in the presence of lead cation (Pb²⁺). The symbol Prerepresents 108-nucleotide precursor DNA (SEQ ID NO 4); Clv,28-nucleotide 5′-cleavage product (SEQ ID NO 5); and M, primer 3a (SEQID NO 6), which corresponds in length to the 5′-cleavage product.

FIG. 3 illustrates the sequence alignment of individual variantsisolated from the population after five rounds of selection. The fixedsubstrate domain is shown at the top, with the target riboadenylateidentified via an inverted triangle. Substrate nucleotides that arecommonly involved in presumed base-pairing interactions are indicated byvertical bars. Sequences corresponding to the 50 initially-randomizednucleotides are aligned antiparallel to the substrate domain. All of thevariants are 3′-terminated by the fixed sequence 5′-CGGTAAGCTTGGCAC-3′(not shown; SEQ ID NO 1). Nucleotides within the initially-randomizedregion that are presumed to form base pairs with the substrate domainare indicated on the right and left sides of the Figure; the putativebase-pair-forming regions of the enzymatic DNA molecules areindividually boxed in each sequence shown. Conserved regions areillustrated via the two large, centrally-located boxes.

FIGS. 4A and 4B illustrate DNA-catalyzed cleavage of an RNA phosphoesterin an intermolecular reaction that proceeds with catalytic turnover.

FIG. 4A is a diagrammatic representation of the complex formed betweenthe 19mer substrate (3′-TCACTATrAGGAAGAGATGG-5′, SEQ ID NO 2) and 38merDNA enzyme (5′-ACACATCTCTGAAGTAGCGCCGCCGTATAGTGACGCTA-3′, SEQ ID NO 3).The substrate contains a single adenosine ribonucleotide (“rA”, adjacentto the arrow), flanked by deoxyribonucleotides. The synthetic DNA enzymeis a 38-nucleotide portion of the most frequently occurring variantshown in FIG. 3. Highly-conserved nucleotides located within theputative catalytic domain are “boxed”. As illustrated, one conservedsequence is “AGCG”, while another is “CG” (reading in the 5′→3′direction).

FIG. 4B shows an Eadie-Hofstee plot used to determine K_(m) (negativeslope) and V_(max) (y-intercept) for DNA-catalyzed cleavage of[5′-³²P]-labeled substrate under conditions identical to those employedduring in vitro selection. Initial rates of cleavage were determined forreactions involving 5 nM DNA enzyme and either 0.125, 0.5, 1, 2, or 4 μMsubstrate.

FIG. 5 is a photographic representation showing a polyacrylamide geldemonstrating specific endoribonuclease activity of four families ofselected catalytic DNAs. Selection of a Pb²⁺-dependent family ofmolecules was repeated in a side-by-side fashion as a control (firstgroup). In the second group, Zn²⁺ is used as the cation; in group three,the cation is Mn²⁺; and in the fourth group, the cation is Mg²⁺. A fifthsite on the gel consists of the cleavage product alone, as a marker.

As noted, there are three lanes within each of the aforementioned fourgroups. In each group of three lanes, the first lane shows the lack ofactivity of the selected population in the absence of the metal cation,the second lane shows the observed activity in the presence of the metalcation, and the third lane shows the lack of activity of the startingpool (G0).

FIGS. 6A and 6B provide two-dimensional illustrations of a “progenitor”catalytic DNA molecule and one of several catalytic DNA moleculesobtained via the selective amplification methods disclosed herein,respectively.

FIG. 6A illustrates an exemplary molecule from the starting pool,showing the overall configuration of the molecules represented by SEQ IDNO 23. As illustrated, various complementary nucleotides flank therandom (N₄₀) region.

FIG. 6B is a diagrammatic representation of one of the Mg²⁺-dependentcatalytic DNA molecules (residue nos. 11-89 of SEQ ID NO 23) (or“DNAzymes”) generated via the within-described procedures. The locationof the ribonucleotide in the substrate nucleic acid is indicated via thearrow in both FIGS. 6A and 6B.

FIG. 7 illustrates some of the results of ten rounds of in vitroselective amplification carried out essentially as described in Example5 hereinbelow. As shown, two sites and two families of catalysts emergedas displaying the most efficient cleavage of the target sequence.Cleavage conditions were essentially as indicated in FIG. 7, namely, 10mM Mg²⁺, pH 7.5, and 37° C.; data collected after the reaction ran for 2hours is shown. Cleavage (%) is shown plotted against the number ofgenerations (here, 0 through 10). The number/prevalence of catalytic DNAmolecules capable of cleaving the target sequence at the indicated sitesin the substrate is illustrated via the vertical bars, with cleavage atG↓UAACUAGAGAU (SEQ ID NO 49) shown by the striped bars, and withcleavage at GUAACUA↓GAGAU (SEQ ID NO 49) illustrated via the open(lightly-shaded) bars.

FIG. 8 illustrates the nucleotide sequences, cleavage sites, andturnover rates of two catalytic DNA molecules of the present invention,clones 8-17 (residue nos. 1-24 of SEQ ID NO 56) and 10-23. Reactionconditions were as shown, namely, 10 mM Mg²⁺, pH 7.5, and 37° C. TheDNAzyme identified as clone 8-17 is illustrated on the left, with thesite of cleavage of the RNA substrate indicated by the arrow. Thesubstrate sequence (5′-GGAAAAAGUAACUAGAGAUGGAAG-3′) (residue nos. 1-34of SEQ ID NO 56)—(residue nos. 1-24 of SEQ ID NO 51)—which is separatefrom the DNAzyme (i.e., intermolecular cleavage is shown)—is labeled assuch. Similarly, the DNAzyme identified herein as 10-23 (residue nos.3-33 of SEQ ID NO 85) is shown on the right, with the site of cleavageof the RNA substrate indicated by the arrow. Again, the substratesequence is indicated. For the 8-17 enzyme, the turnover rate wasapproximately 0.6 hr⁻¹; for the 10-23 enzyme, the turnover rate wasapproximately 1 hr⁻¹. Noncomplementary pairings are indicated with aclosed circle (), whereas complementary pairings are indicated with avertical line (|).

FIG. 9 further illustrates the nucleotide sequences, cleavage sites, andturnover rates of two catalytic DNA molecules of the present invention,clones 8-17 and 10-23. Reaction conditions were as shown, namely, 10 mMMg²⁺, pH 7.5, and 37° C. As in FIG. 8, the DNAzyme identified as clone8-17 (residue nos. 4-30 of SEQ ID NO 56) is illustrated on the left,with the site of cleavage of the RNA substrate indicated by the arrow.The substrate sequence (5′-GGAAAAAGUAACUAGAGAUGGAAG-3′) (residue nos.1-24 of SEQ ID NO 51)—which is separate from the DNAzyme (i.e.,intermolecular cleavage is shown)—is labeled as such. Similarly, theDNAzyme identified herein as 10-23 is shown on the right, with the siteof cleavage of the RNA substrate indicated by the arrow. Again, thesubstrate sequence is indicated. For the 8-17 enzyme, k_(obs) wasapproximately 0.002 min⁻¹; for the 10-23 enzyme, the value of k_(obs)was approximately 0.01 min⁻¹. Noncomplementary pairings are indicatedwith a closed circle (), whereas complementary pairings are indicatedwith a vertical line (|).

DETAILED DESCRIPTION

A. Definitions

As used herein, the term “deoxyribozyme” is used to describe aDNA-containing nucleic acid that is capable of functioning as an enzyme.In the present disclosure, the term “deoxyribozyme” includesendoribonucleases and endodeoxyribonucleases, although deoxyribozymeswith endoribonuclease activity are particularly preferred. Other termsused interchangeably with deoxyribozyme herein are “enzymatic DNAmolecule”, “DNAzyme”, or “catalytic DNA molecule”, which terms shouldall be understood to include enzymatically active portions thereof,whether they are produced synthetically or derived from organisms orother sources.

The term “enzymatic DNA molecules” also includes DNA molecules that havecomplementarity in a substrate-binding region to a specifiedoligonucleotide target or substrate; such molecules also have anenzymatic activity which is active to specifically cleave theoligonucleotide substrate. Stated in another fashion, the enzymatic DNAmolecule is capable of cleaving the oligonucleotide substrateintermolecularly. This complementarity functions to allow sufficienthybridization of the enzymatic DNA molecule to the substrateoligonucleotide to allow the intermolecular cleavage of the substrate tooccur. While one-hundred percent (100%) complementarity is preferred,complementarity in the range of 75-100% is also useful and contemplatedby the present invention.

Enzymatic DNA molecules of the present invention may alternatively bedescribed as having nuclease or ribonuclease activity. These terms maybe used interchangeably herein.

The term “enzymatic nucleic acid” as used herein encompasses enzymaticRNA or DNA molecules, enzymatic RNA-DNA polymers, and enzymaticallyactive portions or derivatives thereof, although enzymatic DNA moleculesare a particularly preferred class of enzymatically active moleculesaccording to the present invention.

The term “endodeoxyribonuclease”, as used herein, is an enzyme capableof cleaving a substrate comprised predominantly of DNA. The term“endoribonuclease”, as used herein, is an enzyme capable of cleaving asubstrate comprised predominantly of RNA.

As used herein, the term “base pair” (bp) is generally used to describea partnership of adenine (A) with thymine (T) or uracil (U), or ofcytosine (C) with guanine (G), although it should be appreciated thatless-common analogs of the bases A, T, C, and G (as well as U) mayoccasionally participate in base pairings. Nucleotides that normallypair up when DNA or RNA adopts a double stranded configuration may alsobe referred to herein as “complementary bases”.

“Complementary nucleotide sequence” generally refers to a sequence ofnucleotides in a single-stranded molecule or segment of DNA or RNA thatis sufficiently complementary to that on another single oligonucleotidestrand to specifically hybridize to it with consequent hydrogen bonding.

“Nucleotide” generally refers to a monomeric unit of DNA or RNAconsisting of a sugar moiety (pentose), a phosphate group, and anitrogenous heterocyclic base. The base is linked to the sugar moietyvia the glycosidic carbon (1′ carbon of the pentose) and thatcombination of base and sugar is a “nucleoside”. When the nucleosidecontains a phosphate group bonded to the 3′ or 5′ position of thepentose, it is referred to as a nucleotide. A sequence of operativelylinked nucleotides is typically referred to herein as a “base sequence”or “nucleotide sequence”, and their grammatical equivalents, and isrepresented herein by a formula whose left to right orientation is inthe conventional direction of 5′-terminus to 3′-terminus, unlessotherwise specified.

“Nucleotide analog” generally refers to a purine or pyrimidinenucleotide that differs structurally from A, T, G, C, or U, but issufficiently similar to substitute for the normal nucleotide in anucleic acid molecule. As used herein, the term “nucleotide analog”encompasses altered bases, different or unusual sugars (i.e. sugarsother than the “usual” pentose), or a combination of the two. A listingof exemplary analogs wherein the base has been altered is provided insection C hereinbelow.

“Oligonucleotide or polynucleotide” generally refers to a polymer ofsingle- or double-stranded nucleotides. As used herein,“oligonucleotide” and its grammatical equivalents will include the fullrange of nucleic acids. An oligonucleotide will typically refer to anucleic acid molecule comprised of a linear strand of ribonucleotides.The exact size will depend on many factors, which in turn depends on theultimate conditions of use, as is well known in the art.

As used herein, the term “physiologic conditions” is meant to suggestreaction conditions emulating those found in mammalian organisms,particularly humans. While variables such as temperature, availabilityof cations, and pH ranges may vary as described in greater detail below,“physiologic conditions” generally comprise a temperature of about35-40° C., with 37° C. being particularly preferred, as well as a pH ofabout 7.0-8.0, with 7.5 being particularly preferred, and furthercomprise the availability of cations, preferably divalent and/ormonovalent cations, with a concentration of about 2-15 mM Mg²⁺ and 0-1.0M Na⁺ being particularly preferred. “Physiologic conditions”, as usedherein, may optionally include the presence of free nucleoside cofactor.As noted previously, preferred conditions are described in greaterdetail below.

B. Enzymatic DNA Molecules

In various embodiments, an enzymatic DNA molecule of the presentinvention may combine one or more modifications or mutations includingadditions, deletions, and substitutions. In alternative embodiments,such mutations or modifications may be generated using methods whichproduce random or specific mutations or modifications. These mutationsmay, for example, change the length of, or alter the nucleotide sequenceof, a loop, a spacer region or the recognition sequence (or domain). Oneor more mutations within one catalytically active enzymatic DNA moleculemay be combined with the mutation(s) within a second catalyticallyactive enzymatic DNA molecule to produce a new enzymatic DNA moleculecontaining the mutations of both molecules.

In other preferred embodiments, an enzymatic DNA molecule of the presentinvention may have random mutations introduced into it using a varietyof methods well known to those skilled in the art. For example, themethods described by Cadwell and Joyce (PCR Methods and Applications 2:28-33 (1992)) are particularly preferred for use as disclosed herein,with some modifications, as described in the Examples that follow. (Alsosee Cadwell and Joyce, PCR Methods and Applications 3 (Suppl.):S136-S140 (1994).) According to this modified PCR method, random pointmutations may be introduced into cloned genes.

The aforementioned methods have been used, for example, to mutagenizegenes encoding ribozymes with a mutation rate of 0.66%±0.13% (95%confidence interval) per position, as determined by sequence analysis,with no strong preferences observed with respect to the type of basesubstitution. This allows the introduction of random mutations at anyposition in the enzymatic DNA molecules of the present invention.

Another method useful in introducing defined or random mutations isdisclosed in Joyce and Inoue, Nucleic Acids Research 17: 711-722 (1989).This latter method involves excision of a template (coding) strand of adouble-stranded DNA, reconstruction of the template strand withinclusion of mutagenic oligonucleotides, and subsequent transcription ofthe partially-mismatched template. This allows the introduction ofdefined or random mutations at any position in the molecule by includingpolynucleotides containing known or random nucleotide sequences atselected positions.

Enzymatic DNA molecules of the present invention may be of varyinglengths and folding patterns, as appropriate, depending on the type andfunction of the molecule. For example, enzymatic DNA molecules may beabout 15 to about 400 or more nucleotides in length, although a lengthnot exceeding about 250 nucleotides is preferred, to avoid limiting thetherapeutic usefulness of molecules by making them too large orunwieldy. In various preferred embodiments, an enzymatic DNA molecule ofthe present invention is at least about 20 nucleotides in length and,while useful molecules may exceed 100 nucleotides in length, preferredmolecules are generally not more than about 100 nucleotides in length.

In various therapeutic applications, enzymatic DNA molecules of thepresent invention comprise the enzymatically active portions ofdeoxyribozymes. In various embodiments, enzymatic DNA molecules of thepresent invention preferably comprise not more than about 200nucleotides. In other embodiments, a deoxyribozyme of the presentinvention comprises not more than about 100 nucleotides. In still otherpreferred embodiments, deoxyribozymes of the present invention are about20-75 nucleotides in length, more preferably about 20-65 nucleotides inlength. Other preferred enzymatic DNA molecules are about 10-50nucleotides in length.

In other applications, enzymatic DNA molecules may assume configurationssimilar to those of “hammerhead” ribozymes. Such enzymatic DNA moleculesare preferably no more than about 75-100 nucleotides in length, with alength of about 20-50 nucleotides being particularly preferred.

In general, if one intends to synthesize molecules for use as disclosedherein, the larger the enzymatic nucleic acid molecule is, the moredifficult it is to synthesize. Those of skill in the art will certainlyappreciate these design constraints. Nevertheless, such larger moleculesremain within the scope of the present invention.

It is also to be understood that an enzymatic DNA molecule of thepresent invention may comprise enzymatically active portions of adeoxyribozyme or may comprise a deoxyribozyme with one or moremutations, e.g., with one or more base-pair-forming sequences or spacersabsent or modified, as long as such deletions, additions ormodifications do not adversely impact the molecule's ability to performas an enzyme.

The recognition domain of an enzymatic DNA molecule of the presentinvention typically comprises two nucleotide sequences flanking acatalytic domain, and typically contains a sequence of at least about 3to about 30 bases, preferably about 6 to about 15 bases, which arecapable of hybridizing to a complementary sequence of bases within thesubstrate nucleic acid giving the enzymatic DNA molecule its highsequence specificity. Modification or mutation of the recognition sitevia well-known methods allows one to alter the sequence specificity ofan enzymatic nucleic acid molecule. (See, e.g., Joyce et al., NucleicAcids Research 17: 711-712 (1989.))

Enzymatic nucleic acid molecules of the present invention also includethose with altered recognition sites or domains. In various embodiments,these altered recognition domains confer unique sequence specificitieson the enzymatic nucleic acid molecule including such recognitiondomains. The exact bases present in the recognition domain determine thebase sequence at which cleavage will take place. Cleavage of thesubstrate nucleic acid occurs within the recognition domain. Thiscleavage leaves a 2′, 3′, or 2′,3′-cyclic phosphate group on thesubstrate cleavage sequence and a 5′ hydroxyl on the nucleotide that wasoriginally immediately 3′ of the substrate cleavage sequence in theoriginal substrate. Cleavage can be redirected to a site of choice bychanging the bases present in the recognition sequence (internal guidesequence). See Murphy et al., Proc. Natl. Acad. Sci. USA 86: 9218-9222(1989).

Moreover, it may be useful to add a polyamine to facilitate recognitionand binding between the enzymatic DNA molecule and its substrate.Examples of useful polyamines include spermidine, putrescine orspermine. A spermidine concentration of about 1 mM may be effective inparticular embodiments, while concentrations ranging from about 0.1 mMto about 10 mM may also be useful.

In various alternative embodiments, an enzymatic DNA molecule of thepresent invention has an enhanced or optimized ability to cleave nucleicacid substrates, preferably RNA substrates. As those of skill in the artwill appreciate, the rate of an enzyme-catalyzed reaction variesdepending upon the substrate and enzyme concentrations and, in general,levels off at high substrate or enzyme concentrations. Taking sucheffects into account, the kinetics of an enzyme-catalyzed reaction maybe described in the following terms, which define the reaction.

The enhanced or optimized ability of an enzymatic DNA molecule of thepresent invention to cleave an RNA substrate may be determined in acleavage reaction with varying amounts of labeled RNA substrate in thepresence of enzymatic DNA molecule. The ability to cleave the substrateis generally defined by the catalytic rate (k_(cat)) divided by theMichaelis constant (K_(M)). The symbol k_(cat) represents the maximalvelocity of an enzyme reaction when the substrate approaches asaturation value. K_(M) represents the substrate concentration at whichthe reaction rate is one-half maximal.

For example, values for K_(M) and k_(cat) may be determined in thisinvention by experiments in which the substrate concentration [S] is inexcess over enzymatic DNA molecule concentration [E]. Initial rates ofreaction (v₀) over a range of substrate concentrations are estimatedfrom the initial linear phase, generally the first 5% or less of thereaction. Data points are fit by a least squares method to a theoreticalline given by the equation: v=−K_(M)(v₀/[S])+V_(max). Thus, k_(cat) andK_(M) are determined by the initial rate of reaction, v₀, and thesubstrate concentration [S].

In various alternative embodiments, an enzymatic DNA molecule of thepresent invention has an enhanced or optimized ability to cleave nucleicacid substrates, preferably RNA substrates. In preferred embodiments,the enhanced or optimized ability of an enzymatic DNA molecule to cleaveRNA substrates shows about a 10- to 10⁹-fold improvement over theuncatalyzed rate. In more preferred embodiments, an enzymatic DNAmolecule of the present invention is able to cleave RNA substrates at arate that is about 10³- to 10⁷-fold improved over “progenitor” species.In even more preferred embodiments, the enhanced or optimized ability tocleave RNA substrates is expressed as a 10⁴- to 10⁶-fold improvementover the progenitor species. One skilled in the art will appreciate thatthe enhanced or optimized ability of an enzymatic DNA molecule to cleavenucleic acid substrates may vary depending upon the selectionconstraints applied during the in vitro evolution procedure of theinvention.

Various preferred methods of modifying deoxyribozymes and otherenzymatic DNA molecules and nucleases of the present invention arefurther described in Examples 1-3 hereinbelow.

C. Nucleotide Analogs

As noted above, the term “nucleotide analog” as used herein generallyrefers to a purine or pyrimidine nucleotide that differs structurallyfrom A, T, G, C, or U, but is sufficiently similar to substitute forsuch “normal” nucleotides in a nucleic acid molecule. As used herein,the term “nucleotide analog” encompasses altered bases, different (orunusual) sugars, altered phosphate backbones, or any combination ofthese alterations. Examples of nucleotide analogs useful according tothe present invention include those listed in the following Table, mostof which are found in the approved listing of modified bases at 37 CFR§1.822 (which is incorporated herein by reference).

TABLE 1 Nucleotide Analogs Abbreviation Description ac4c4-acetylcytidine chm5u 5-(carboxyhydroxylmethyl)uridine cm2′-O-methylcytidine cmnm5s2u 5-carboxymethylaminomethyl-2-thiouridine ddihydrouridine fm 2′-O-methylpseudouridine galq β, D-galactosylqueosinegm 2′-O-methylguanosine l inosine i6a N6-isopentenyladenosine m1a1-methyladenosine m1f 1-methylpseudouridine m1g 1-methylguanosine ml11-methylinosine m22g 2,2-dimethylguanosine m2a 2-methyladenosine m2g2-methylguanosine m3c 3-methylcytidine m5c 5-methylcytidine m6aN6-methyladenosine m7g 7-methylguanosine mam5u5-methylaminomethyluridine mam5s2u 5-methoxyaminomethyl-2-thiouridinemanq β, D-mannosylmethyluridine mcm5s2u 5-methoxycarbonylmethyluridinemo5u 5-methoxyuridine ms2i6a 2-methylthio-N6-isopentenyladenosine ms2t6aN-((9-β-D-ribofuranosyl-2-methylthiopurine-6- yl)carbamoyl)threoninemt6a N-((9-β-D-ribofuranosylpurine-6-yl)N-methyl- carbamoyl)threonine mvuridine-5-oxyacetic acid methylester o5u uridine-5-oxyacetic acid (v)osyw wybutoxosine p pseudouridine q queosine s2c 2-thiocytidine s2t5-methyl-2-thiouridine s2u 2-thiouridine s4u 4-thiouridine t5-methyluridine t6aN-((9-β-D-ribofuranosylpurine-6-yl)carbamoyl)threoninetm2′-O-methyl-5-methyluridine um 2′-O-methyluridine yw wybutosine x3-(3-amino-3-carboxypropyl)uridine, (acp3)u araU β, D-arabinosyl araT β,D-arabinosyl

Other useful analogs include those described in published internationalapplication no. WO 92/20823 (the disclosures of which are incorporatedherein by reference), or analogs made according to the methods disclosedtherein. Analogs described in DeMesmaeker, et al., Angew. Chem. Int. Ed.Engl. 33: 226-229 (1994); DeMesmaeker, et al., Synlett: 733-736 (October1993); Nielsen, et al., Science 254: 1497-1500 (1991); and Idziak, etal., Tetrahedron Letters 34: 5417-5420 (1993) are also useful accordingto the within-disclosed invention and said disclosures are incorporatedby reference herein.

D. Methods of Engineering Enzymatic DNA Molecules

The present invention also contemplates methods of producing nucleicacid molecules having a predetermined activity. In one preferredembodiment, the nucleic acid molecule is an enzymatic DNA molecule. Inanother variation, the desired activity is a catalytic activity.

In one embodiment, the present invention contemplates methods ofsynthesizing enzymatic DNA molecules that may then be “engineered” tocatalyze a specific or predetermined reaction. Methods of preparingenzymatic DNA molecules are described herein; see, e.g., Examples 1-3hereinbelow. In other embodiments, an enzymatic DNA molecule of thepresent invention may be engineered to bind small molecules or ligands,such as adenosine triphosphate (ATP). (See, e.g., Sassanfar, et al.,Nature 364: 550-553 (1993).)

In another embodiment, the present invention contemplates that apopulation of enzymatic DNA molecules may be subjected to mutagenizingconditions to produce a diverse population of mutant enzymatic DNAmolecules (which may alternatively be called “deoxyribozymes” or“DNAzymes”). Thereafter, enzymatic DNA molecules having desiredcharacteristics are selected and/or separated from the population andare subsequently amplified.

Alternatively, mutations may be introduced in the enzymatic DNA moleculeby altering the length of the recognition domains of the enzymatic DNAmolecule. The recognition domains of the enzymatic DNA moleculeassociate with a complementary sequence of bases within a substratenucleic acid sequence. Methods of altering the length of the recognitiondomains are known in the art and include PCR, for example; usefultechniques are described further in the Examples below.

Alteration of the length of the recognition domains of an enzymatic DNAmolecule may have a desirable effect on the binding specificity of theenzymatic DNA molecule. For example, an increase in the length of therecognition domains may increase binding specificity between theenzymatic DNA molecule and the complementary base sequences of anoligonucleotide in a substrate, or may enhance recognition of aparticular sequence in a hybrid substrate. In addition, an increase inthe length of the recognition domains may also increase the affinitywith which it binds to substrate. In various embodiments, these alteredrecognition domains in the enzymatic DNA molecule confer increasedbinding specificity and affinity between the enzymatic DNA molecule andits substrate.

It has recently been noted that certain oligonucleotides are able torecognize and bind molecules other than oligonucleotides withcomplementary sequences. These oligonucleotides are often given the name“aptamers”. For example, Ellington and Szostak describe RNA moleculesthat are able to bind a variety of organic dyes (Nature 346: 818-822(1990)), while Bock, et al. describe ssDNA molecules that bind humanthrombin (Nature 355: 564-566 (1992)). Similarly, Jellinek, et al.describe RNA ligands to basic fibroblast growth factor (PNAS USA 90:11227-11231 (1993)). Thus, it is further contemplated herein that thecatalytically active DNA enzymes of the present invention may beengineered according to the within-described methods to display avariety of capabilities typically associated with aptamers.

One of skill in the art should thus appreciate that the enzymatic DNAmolecules of this invention can be altered at any nucleotide sequence,such as the recognition domains, by various methods disclosed herein,including PCR and 3SR (self-sustained sequence replication—see Example 1below). For example, additional nucleotides can be added to the 5′ endof the enzymatic DNA molecule by including additional nucleotides in theprimers.

Enzymatic DNA molecules of the present invention may also be prepared orengineered in a more non-random fashion via use of methods such assite-directed mutagenesis. For example, site-directed mutagenesis may becarried out essentially as described in Morinaga, et al., Biotechnology2: 636 (1984), modified as described herein, for application todeoxyribozymes. Useful methods of engineering enzymatic DNA moleculesare further described in the Examples below.

In one disclosed embodiment, an enzymatic DNA molecule of the presentinvention comprises a conserved core flanked by two substrate binding(or recognition) domains or sequences that interact with the substratethrough base-pairing interactions. In various embodiments, the conservedcore comprises one or more conserved domains or sequences. In anothervariation, an enzymatic DNA molecule further comprises a “spacer” region(or sequence) between the regions (or sequences) involved in basepairing. In still another variation, the conserved core is “interrupted”at various intervals by one or more less-conserved variable or “spacer”nucleotides.

In various embodiments, the population of enzymatic DNA molecules ismade up of at least 2 different types of deoxyribozyme molecules. Forexample, in one variation, the molecules have differing sequences. Inanother variation, the deoxyribozymes are nucleic acid molecules havinga nucleic acid sequence defining a recognition domain that is contiguousor adjacent to the 5′-terminus of the nucleotide sequence. In variousalternative embodiments, enzymatic DNA molecules of the presentinvention may further comprise one or more spacer regions located3′-terminal to the recognition domains, one or more loops located3′-terminal to the recognition domains and/or spacer regions. In othervariations, a deoxyribozyme of the present invention may comprise one ormore regions which are capable of hybridizing to other regions of thesame molecule. Other characteristics of enzymatic DNA molecules producedaccording to the presently-disclosed methods are described elsewhereherein.

In other embodiments, mutagenizing conditions include conditions thatintroduce either defined or random nucleotide substitutions within anenzymatic DNA molecule. Examples of typical mutagenizing conditionsinclude conditions disclosed in other parts of this specification andthe methods described by Joyce et al., Nucl. Acids Res. 17: 711-722(1989); Joyce, Gene 82: 83-87(1989); and Beaudry and Joyce, Science 257:635-41 (1992).

In still other embodiments, a diverse population of mutant enzymaticnucleic acid molecules of the present invention is one that contains atleast 2 nucleic acid molecules that do not have the exact samenucleotide sequence. In other variations, from such a diversepopulation, an enzymatic DNA molecule or other enzymatic nucleic acidhaving a predetermined activity is then selected on the basis of itsability to perform the predetermined activity. In various embodiments,the predetermined activity comprises, without limitation, enhancedcatalytic activity, decreased K_(M), enhanced substrate binding ability,altered substrate specificity, and the like.

Other parameters which may be considered aspects of enzyme performanceinclude catalytic activity or capacity, substrate binding ability,enzyme turnover rate, enzyme sensitivity to feedback mechanisms, and thelike. In certain aspects, substrate specificity may be considered anaspect of enzyme performance, particularly in situations in which anenzyme is able to recognize and bind two or more competing substrates,each of which affects the enzyme's performance with respect to the othersubstrate(s).

Substrate specificity, as used herein, may refer to the specificity ofan enzymatic nucleic acid molecule as described herein for a particularsubstrate, such as one comprising ribonucleotides only,deoxyribonucleotides only, or a composite of both. Substrate moleculesmay also contain nucleotide analogs. In various embodiments, anenzymatic nucleic acid molecule of the present invention maypreferentially bind to a particular region of a hybrid or non-hybridsubstrate.

The term or parameter identified herein as “substrate specificity” mayalso include sequence specificity; i.e., an enzymatic nucleic acidmolecule of the present invention may “recognize” and bind to a nucleicacid substrate having a particular nucleic acid sequence. For example,if the substrate recognition domains of an enzymatic nucleic acidmolecule of the present invention will only bind to substrate moleculeshaving a series of one or two ribonucleotides (e.g., rA) in a row, thenthe enzymatic nucleic acid molecule will tend not to recognize or bindnucleic acid substrate molecules lacking such a sequence.

With regard to the selection process, in various embodiments, selectingincludes any means of physically separating the mutant enzymatic nucleicacids having a predetermined activity from the diverse population ofmutant enzymatic nucleic acids. Often, selecting comprises separation bysize, by the presence of a catalytic activity, or by hybridizing themutant nucleic acid to another nucleic acid, to a peptide, or some othermolecule that is either in solution or attached to a solid matrix.

In various embodiments, the predetermined activity is such that themutant enzymatic nucleic acid having the predetermined activity becomeslabeled in some fashion by virtue of the activity. For example, thepredetermined activity may be an enzymatic DNA molecule activity wherebythe activity of the mutant enzymatic nucleic acid upon its substratecauses the mutant enzymatic nucleic acid to become covalently linked toit. The mutant enzymatic nucleic acid is then selected by virtue of thecovalent linkage.

In other embodiments, selecting a mutant enzymatic nucleic acid having apredetermined activity includes amplification of the mutant enzymaticnucleic acid (see, e.g., Joyce, Gene 82: 8:3-87 (1989); Beaudry andJoyce, Science 257: 635-41 (1992)). Other methods of selecting anenzymatic nucleic acid molecule having a predetermined characteristic oractivity are described in the Examples section.

E. Compositions

The invention also contemplates compositions containing one or moretypes or populations of enzymatic DNA molecules of the presentinvention; e.g., different types or populations may recognize and cleavedifferent nucleotide sequences. Compositions may further include aribonucleic acid-containing substrate. Compositions according to thepresent invention may further comprise lead ion, magnesium ion, or otherdivalent or monovalent cations, as discussed herein.

Preferably, the anzymatic DNA molecule is present at a concentration ofabout 0.05 μM to about 2 μM. Typically, the enzymatic DNA molecule ispresent at a concentration ratio of enzymatic DNA molecule to substrateof from about 1:5 to about 1:50. More preferably, the enzymatic DNAmolecule is present in the composition at a concentration of about 0.1μM to about 1 μM. Even more preferably, compositions contain theenzymatic DNA molecule at a concentration of about 0.1 μM to about 0.5μM. Preferably, the substrate is present in the composition at aconcentration of about 0.5 μM to about 1000 μM.

One skilled in the art will understand that there are many sources ofnucleic acid-containing substrates including naturally-occurring andsynthetic sources. Sources of suitable substrates include, withoutlimitation, a variety of viral and retroviral agents, including HIV-1,HIV-2, HTLV-I, and HTLV-II.

Other suitable substrates include, without limitation, viral andretroviral agents including those comprising or produced bypicornaviruses, hepadnaviridae (e.g., HBV, HCV), papillomaviruses (e.g.,HPV), gammaherpesvirinae (e.g., EBV), lymphocryptoviruses, leukemiaviruses (e.g., HTLV-I and -II), flaviviruses, togaviruses, herpesviruses(including alphaherpesviruses and betaherpesviruses), cytomegaloviruses(CMV), influenza viruses, and viruses and retroviruses contributing toimmunodeficiency diseases and syndromes (e.g., HIV-1 and -2). Inaddition, suitable substrates include viral and retroviral agents whichinfect non-human primates and other animals including, withoutlimitation, the simian and feline immunodeficiency viruses and bovineleukemia viruses.

Magnesium ion, lead ion, or another suitable monovalent or divalentcation, as described previously, may also be present in the composition,at a concentration ranging from about 1-100 mM. More preferably, thepreselected ion is present in the composition at a concentration ofabout 2 mM to about 50 mM, with a concentration of about 5 mM beingparticularly preferred. One skilled in the art will understand that the10 ion concentration is only constrained by the limits of solubility ofits source (e.g. magnesium) in aqueous solution and a desire to have theenzymatic DNA molecule present in the same composition in an activeconformation.

The invention also contemplates compositions containing an enzymatic DNAmolecule of the present invention, hybriddeoxyribonucleotide-ribonucleotide molecules, and magnesium or lead ionin concentrations as described hereinabove. As noted previously, othermonovalent or divalent ions (e.g., Ca²⁺) may be used in place ofmagnesium.

Also contemplated by the present invention are compositions containingan enzymatic DNA molecule of the present invention, nucleicacid-containing substrate (e.g. RNA), and a preselected ion at aconcentration of greater than about 1 millimolar, wherein said substrateis greater in length than the recognition domains present on theenzymatic DNA molecule.

In one variation, a composition comprises an enzymatic DNAmolecule-substrate complex, wherein base pairing between an enzymaticDNA molecule and its substrate is contiguous. In another embodiment,base pairing between an enzymatic DNA molecule and its substrate isinterrupted by one or more noncomplementary pairs. In a variety ofalternative embodiments, a composition of the present invention mayfurther comprise a monovalent cation, a divalent cation, or both.

In another variation, an enzymatic DNA molecule of the present inventionis capable of functioning efficiently in the presence or absence of adivalent cation. In one variation, a divalent cation is present andcomprises Pb²⁺, Mg²⁺, Mn²⁺, Zn²⁺, or Ca²⁺. Alternatively, an enzymaticDNA molecule of the present invention is capable of functioningefficiently in the presence or absence of monovalent cations. It isanticipated that monovalent or divalent cation concentrations similar tothose described herein for Pb² ⁺ or Mg²⁺ will be useful as disclosedherein.

Optionally, monovalent cations may also be present in addition to, or as“alternatives” for, divalent cations. For example, monovalent cationssuch as sodium (Na⁺) or potassium (K⁺) may be present, either asdissociated ions or in the form of dissociable compounds such as NaCl orKCl.

In one embodiment, the concentration of monovalent cation present in thecomposition ranges from 0-1.0 M. In another embodiment, a monovalentcation is present in a concentration ranging from about 0-200 mM. Inother embodiments, monovalent cations are present in a concentrationranging from about 1-100 mM. Alternatively, the concentration ofmonovalent cations ranges from about 2 mM-50 mM. In still otherembodiments, the concentration ranges from about 2 mM-25 mM.

F. Methods of Using Enzymatic DNA Molecules

The methods of using enzymatic DNA molecules as disclosed herein arelegion. As discussed previously, molecules capable of cleaving the bondslinking neighboring nucleic acids (e.g., phosphoester bonds) havenumerous uses encompassing a wide variety of applications. For example,enzymatic DNA molecules having the within-disclosed capabilities,structures, and/or functions are useful in pharmaceutical and medicalproducts (e.g., for wound debridement, clot dissolution, etc.), as wellas in household items (e.g., detergents, dental hygiene products, meattenderizers). Industrial utility of the within-disclosed compounds,compositions and methods is also contemplated and well within the scopeof the present invention.

The present invention also describes useful methods for cleaving anysingle-stranded, looped, partially or fully double-stranded nucleicacid; the majority of these methods employ the novel enzymaticallyactive nucleic acid molecules of the present invention. In variousembodiments, the single-stranded nucleic acid segment or portion of thesubstrate (or the entire substrate itself) comprises DNA, modified DNA,RNA, modified RNA, or composites thereof. Preferably, the nucleic acidsubstrate need only be single-stranded at or near the substrate cleavagesequence so that an enzymatic nucleic acid molecule of the presentinvention can hybridize to the substrate cleavage sequence by virtue ofthe enzyme's recognition sequence.

A nucleic acid substrate that can be cleaved by a method of thisinvention may be chemically synthesized or enzymatically produced, or itmay be isolated from various sources such as phages, viruses,prokaryotic cells, or eukaryotic cells, including animal cells, plantcells, yeast cells and bacterial cells. Chemically synthesized single-and double-stranded nucleic acids are commercially available from manysources including, without limitation, Research Genetics (Huntsville,Ala.).

RNA substrates may also be synthesized using an Applied Biosystems(Foster City, Calif.) oligonucleotide synthesizer according to themanufacturer's instructions. Single-stranded phage are also a source ofnucleic acid substrates. (See, e.g., Messing et al., PNAS USA 74:3642-3646 (1977), and Yanisch-Perron et al., Gene 33: 103-119 (1985).)Bacterial cells containing single-stranded phage would also be a readysource of suitable single-stranded nucleic acid substrates.

Single-stranded RNA cleavable by a method of the present invention couldbe provided by any of the RNA viruses such as the picornaviruses,togaviruses, orthomyxoviruses, pararnyxoviruses, rhabdoviruses,coronaviruses, arenaviruses or retroviruses. As noted previously, a widevariety of prokaryotic and eukaryotic cells may also be excellentsources of suitable nucleic acid substrates.

The methods of this invention may be used on single-stranded nucleicacids or single-stranded portions of looped or double-stranded nucleicacids that are present inside a cell, including eukaryotic, procaryotic,plant, animal, yeast or bacterial cells. Under these conditions anenzymatic nucleic acid molecule (e.g., an enzymatic DNA molecule ordeoxyribozyme) of the present invention could act as an anti-viral agentor a regulator of gene expression. Examples of such uses of enzymaticDNA molecules of the present invention are described furtherhereinbelow.

In the majority of methods of the present invention, cleavage ofsingle-stranded nucleic acids occurs at the 3′-terminus of apredetermined base sequence. This predetermined base sequence orsubstrate cleavage sequence typically contains from 1 to about 10nucleotides. In other preferred embodiments, an enzymatic DNA moleculeof the present invention is able to recognize nucleotides eitherupstream, or upstream and downstream of the cleavage site. In variousembodiments, an enzymatic DNA molecule is able to recognize about 2-10nucleotides upstream of the cleavage site; in other embodiments, anenzymatic DNA molecule is able to recognize about 2-10 nucleotidesupstream and about 2-10 nucleotides downstream of the cleavage site.Other preferred embodiments contemplate an enzymatic DNA molecule thatis capable of recognizing a nucleotide sequence up to about 30nucleotides in length, with a length up to about 20 nucleotides beingeven more preferred.

The within-disclosed methods allow cleavage at any nucleotide sequenceby altering the nucleotide sequence of the recognition domains of theenzymatic DNA molecule. This allows cleavage of single-stranded nucleicacid in the absence of a restriction endonuclease site at the selectedposition.

An enzymatic DNA molecule of the present invention may be separated fromany portion of the single-stranded nucleic acid substrate that remainsattached to the enzymatic DNA molecule by site-specific hydrolysis atthe appropriate cleavage site. Separation of the enzymatic DNA moleculefrom the substrate (or “cleavage product”) allows the enzymatic DNAmolecule to carry out another cleavage reaction.

Generally, the nucleic acid substrate is treated under appropriatenucleic acid cleaving conditions—preferably, physiologic conditions—withan effective amount of an enzymatic DNA molecule of the presentinvention. If the nucleic acid substrate comprises DNA, cleavingconditions may include the presence of a divalent cation at aconcentration of about 2-10 mM.

An effective amount of an enzymatic DNA molecule is the amount requiredto cleave a predetermined base sequence present within thesingle-stranded nucleic acid. Preferably, the enzymatic DNA molecule ispresent at a molar ratio of DNA molecule to substrate cleavage sites of1 to 20. This ratio may vary depending on the length of treating andefficiency of the particular enzymatic DNA molecule under the particularnucleic acid cleavage conditions employed.

Thus, in one preferred embodiment, treating typically involves admixing,in aqueous solution, the RNA-containing substrate and the enzyme to forma cleavage admixture, and then maintaining the admixture thus formedunder RNA cleaving conditions for a time period sufficient for theenzymatic DNA molecule to cleave the RNA substrate at any of thepredetermined nucleotide sequences present in the RNA. In variousembodiments, a source of ions is also provided—i.e. monovalent ordivalent cations, or both.

In one embodiment of the present invention, the amount of time necessaryfor the enzymatic DNA molecule to cleave the single-stranded nucleicacid has been predetermined. The amount of time is from about 1 minuteto about 24 hours and will vary depending upon the concentration of thereactants and the temperature of the reaction. Usually, this time periodis from about 10 minutes to about 2 hours such that the enzymatic DNAmolecule cleaves the single-stranded nucleic acid at any of thepredetermined nucleotide sequences present.

The invention further contemplates that the nucleic acid cleavingconditions include the presence of a source of divalent cations (e.g.,PbOAc) at a concentration of about 2-100 mM. Typically, the nucleic acidcleaving conditions include divalent cation at a concentration of about2 mM to about 10 mM, with a concentration of about 5 mM beingparticularly preferred.

The optimal cationic concentration to include in the nucleic acidcleaving conditions can be easily determined by determining the amountof single-stranded nucleic acid cleaved at a given cation concentration.One skilled in the art will understand that the optimal concentrationmay vary depending on the particular enzymatic DNA molecule employed.

The present invention further contemplates that the nucleic acidcleaving conditions include a pH of about pH 6.0 to about pH 9.0. In onepreferred embodiment, the pH ranges from about pH 6.5 to pH 8.0. Inanother preferred embodiment, the pH emulates physiological conditions,i.e., the pH is about 7.0-7.8, with a pH of about 7.5 being particularlypreferred.

One skilled in the art will appreciate that the methods of the presentinvention will work over a wide pH range so long as the pH used fornucleic acid cleaving is such that the enzymatic DNA molecule is able toremain in an active conformation. An enzymatic DNA molecule in an activeconformation is easily detected by its ability to cleave single-strandednucleic acid at a predetermined nucleotide sequence.

In various embodiments, the nucleic acid cleaving conditions alsoinclude a variety of temperature ranges. As noted previously,temperature ranges consistent with physiological conditions areespecially preferred, although temperature ranges consistent withindustrial applications are also contemplated herein. In one embodiment,the temperature ranges from about 15° C. to about 60° C. In anothervariation, the nucleic acid cleaving conditions include a temperatureranging from about 30° C. to about 56° C. In yet another variation,nucleic acid cleavage conditions include a temperature from about 35° C.to about 50° C. In a preferred embodiment, nucleic acid cleavageconditions comprise a temperature range of about 37° C. to about 42° C.The temperature ranges consistent with nucleic acid cleaving conditionsare constrained only by the desired cleavage rate and the stability ofthat particular enzymatic DNA molecule at that particular temperature.

In various methods, the present invention contemplates nucleic acidcleaving conditions including the presence of a polyamine. Polyaminesuseful for practicing the present invention include spermidine,putrescine, spermine and the like. In one variation, the polyamine ispresent at a concentration of about 0.01 mM to about 10 mM. In anothervariation, the polyamine is present at a concentration of about 1 mM toabout 10 mM. Nucleic acid cleavage conditions may also include thepresence of polyamine at a concentration of about 2 mM to about 5 mM. Invarious preferred embodiments, the polyamine is spermidine.

G. Vectors

The present invention also features expression vectors including anucleic acid segment encoding an enzymatic DNA molecule of the presentinvention situated within the vector, preferably in a manner whichallows expression of that enzymatic DNA molecule within a target cell(e.g., a plant or animal cell).

Thus, in general, a vector according to the present invention preferablyincludes a plasmid, cosmid, phagemid, virus, or phage vector.Preferably, suitable vectors comprise single-stranded DNA (ssDNA)—e.g.,circular phagemid ssDNA. It should also be appreciated that usefulvectors according to the present invention need not be circular.

In one variation, nucleotide sequences flanking each of the additionalenzymatic DNA molecule-encoding sequences are preferably provided, whichsequences may be recognized by the first enzymatic DNA molecule. Theintervening or flanking sequences preferably comprise at least 1nucleotide; more preferably, intervening or flanking sequences are about2-20 nucleotides in length, with sequences of about 5-10 nucleotides inlength being particularly preferred.

The addition of polynucleotide tails may also be useful to protect the3′ end of an enzymatic DNA molecule according to the present invention.These may be provided by attaching a polymeric sequence by employing theenzyme terminal transferase.

A vector according to the present invention includes two or moreenzymatic DNA molecules. In one embodiment, a first enzymatic DNAmolecule has intramolecular cleaving activity and is able to recognizeand cleave nucleotide sequences to release other enzymatic DNAsequences; i.e., it is able to function to “release” other enzymatic DNAmolecules from the vector. For example, a vector is preferablyconstructed so that when the first enzymatic DNA molecule is expressed,that first molecule is able to cleave nucleotide sequences flankingadditional nucleotide sequences encoding a second enzymatic DNAmolecule, a third enzymatic DNA molecule, and so forth. Presuming saidfirst enzymatic DNA molecule (i.e., the “releasing” molecule) is able tocleave oligonucleotide sequences intramolecularly, the additional (e.g.second, third, and so on) enzymatic DNA molecules (i.e., the “released”molecules) need not possess characteristics identical to the “releasing”molecule. For example, in one embodiment, the “released” (i.e., thesecond, third, etc.) enzymatic DNA molecules are able to cleave specificRNA sequences, while the first (“releasing”) enzymatic DNA molecule hasnuclease activity allowing it to liberate the “released” molecules. Inanother embodiment, the “released” enzymatic DNA molecule has amidebond-cleaving activity, while the first (“releasing”) enzymatic DNAmolecule has nuclease activity.

Alternatively, the first enzymatic DNA molecule may be encoded on aseparate vector from the second (and third, fourth, etc.) enzymatic DNAmolecules) and may have intermolecular cleaving activity. As notedherein, the first enzymatic DNA molecule can be a self-cleavingenzymatic DNA molecule (e.g., a deoxyribozyme), and the second enzymaticDNA molecule may be any desired type of enzymatic DNA molecule. When avector is caused to express DNA from these nucleic acid sequences, thatDNA has the ability under appropriate conditions to cleave each of theflanking regions, thereby releasing one or more copies of the secondenzymatic DNA molecule. If desired, several different second enzymaticDNA molecules can be placed in the same cell or carrier to producedifferent deoxyribozymes. It is also contemplated that any one or morevectors may comprise one or more ribozymes or deoxyribozymes in anycombination of “releasing” and “released” enzymatic nucleic acidmolecules, as long as such a combination achieves the desired result:the release of enzymatic nucleic acid molecules that are capable ofcleaving predetermined nucleic acid sequences.

Methods of isolating and purifying enzymatic DNA molecules of thepresent invention are also contemplated. In addition to the methodsdescribed herein, various purification methods (e.g. those using HPLC)and chromatographic isolation techniques are available in the art. See,e.g., the methods described in published international application no.WO 93/23569, the disclosures of which are incorporated herein byreference.

It should also be understood that various combinations of theembodiments described herein are included within the scope of thepresent invention. Other features and advantages of the presentinvention will be apparent from the descriptions hereinabove, from theExamples to follow, and from the claims.

EXAMPLES

The following examples illustrate, but do not limit, the presentinvention.

Example 1 In Vitro Evolution of Enzymatic DNA Molecules: an Overview

In vitro selection and in vitro evolution techniques allow new catalyststo be isolated without a prior knowledge of their composition orstructure. Such methods have been used to obtain RNA enzymes with novelcatalytic properties. For example, ribozymes that undergo autolyticcleavage with lead cation have been derived from a randomized pool oftRNA^(Phe) molecules (Pan and Uhlenbeck, Biochemistry 31: 3887-3895(1992)). Group I ribozyme variants have been isolated that can cleaveDNA (Beaudry and Joyce, Science 257: 635-641 (1992)) or that havealtered metal dependence (Lehman and Joyce, Nature 361: 182-185 (1993)).Starting with a pool of random RNA sequences, molecules have beenobtained that catalyze a polymerase-like reaction (Bartel and Szostak,Science 261: 1411-1418 (1993)). In the present example, refinement ofspecific catalytic properties of an evolved enzyme via alteration of theselection constraints during an in vitro evolution procedure isdescribed.

Darwinian evolution requires the repeated operation of three processes:(a) introduction of genetic variation; (b) selection of individuals onthe basis of some fitness criterion; and (c) amplification of theselected individuals. Each of these processes can be realized in vitro(Joyce, Gene 82: 83 (1989)). A gene can be mutagenized by chemicalmodification, incorporation of randomized mutagenicoligodeoxynucleotides, or inaccurate copying by a polymerase. (See,e.g., Cadwell and Joyce, in PCR Methods and Applications 2: 28-33(1992); Cadwell and Joyce, PCR Methods and Applications 3 (Suppl.):S136-S140 (1994); Chu, et al., Virology 98: 168 (1979); Shortle, et al.,Meth. Enzymol. 100: 457 (1983); Myers, et al., Science 229: 242 (1985);Matteucci, et al., Nucleic Acids Res. 11: 3113 (1983); Wells, et al.,Gene 34: 315 (1985); McNeil, et al., Mol. Cell. Biol. 5: 3545 (1985);Hutchison, et al., PNAS USA 83: 710 (1986); Derbyshire, et al., Gene 46:145 (1986); Zakour, et al., Nature 295: 708 (1982); Lehtovaara, et al.,Protein Eng. 2: 63 (1988); Leung, et al., Technique 1: 11 (1989); Zhou,et al., Nucl. Acids Res. 19: 6052 (1991).)

The gene product can be selected, for example, by its ability to bind aligand or to carry out a chemical reaction. (See, e.g., Joyce, Id.(1989); Robertson and Joyce, Nature 344: 467 (1990); Tuerk, et al.,Science 249: 505 (1990).) The gene that corresponds to the selected geneproduct can be amplified by a reciprocal primer method, such as thepolymerase chain reaction (PCR). (See, e.g., Saiki, et al., Science 230:1350-54 (1985); Saiki, et al., Science 239: 487-491 (1988).)

Alternatively, nucleic acid amplification may be carried out usingself-sustained sequence replication (3SR). (See, e.g., Guatelli, et al.,PNAS USA 87: 1874 (1990), the disclosures of which are incorporated byreference herein.) According to the 3SR method, target nucleic acidsequences may be amplified (replicated) exponentially in vitro underisothermal conditions by using three enzymatic activities essential toretroviral replication: (1) reverse transcriptase, (2) RNase H, and (3)a DNA-dependent RNA polymerase. By mimicking the retroviral strategy ofRNA replication by means of cDNA intermediates, this reactionaccumulates cDNA and RNA copies of the original target.

In summary, if one is contemplating the evolution of a population ofenzymatic DNA molecules, a continuous series of reverse transcriptionand transcription reactions replicates an RNA target sequence by meansof cDNA intermediates. The crucial elements of this design are (a) theoligonucleotide primers both specify the target and contain 5′extensions encoding the T7 RNA polymerase binding site, so that theresultant cDNAs are competent transcription templates; (b) cDNAsynthesis can proceed to completion of both strands due to thedegradation of template RNA in the intermediate RNA-DNA hybrid by RNaseH; and (c) the reaction products (cDNA and RNA) can function astemplates for subsequent steps, enabling exponential replication.

If one is evolving enzymatic DNA molecules, various critical elements ofthis design are somewhat different, as disclosed in these Examples. Forinstance, (1) the oligonucleotide primers specify the target and arepreferably “marked” or labeled in some fashion—e.g., viabiotinylation—so the resultant competent template strands are easilyidentified; and (2) the in vitro selection procedure used preferablydepends upon the identification of the most favorable release mechanism.

A major obstacle to realizing Darwinian evolution in vitro is the needto integrate mutation and amplification, both of which aregenotype-related, with selection, which is phenotype-related. In thecase of nucleic acid enzymes, for which genotype and phenotype areembodied in the same molecule, the task is simplified.

A. Design of Enzymatic DNA Molecules

It is well known that single-stranded DNA can assume interestingtertiary structures. The structure of a “tDNA”, for example, closelyresembles that of the corresponding tRNA. (See Paquette, et al., Eur. J.Biochem. 189: 259-265 (1990).) Furthermore, it has been possible toreplace as many as 31 of 35 ribonucleotides within a hammerheadribozyme, while retaining at least some catalytic activity. (SeePerreault, et al., Nature 344: 565-567 (1990); Williams, et al., Proc.Natl. Acad. Sci. USA 89: 918-921 (1992); Yang, et al., Biochemistry 31:5005-5009 (1992).)

In vitro selection techniques have been applied to large populations ofrandom-sequence DNAs, leading to the recovery of specific DNA “aptamers”that bind a target ligand with high affinity (Bock, et al., Nature 355:564-566 (1992); Ellington & Szostak, Nature 355: 850-852 (1992); Wyatt &Ecker, PNAS USA 91: 1356-1360 (1994)). Recently, two groups carried outthe first NMR structural determination of an aptamer, a 15mer DNA thatforms a G-quartet structure and binds the protein thrombin with highaffinity (Wang, et al., Biochemistry 32: 1899-1904 (1993); Macaya, etal., PNAS USA 90: 3745-3749 (1993)). These findings were corroborated byan X-ray crystallographic analysis (Padmanabhan, et al., J. Biol. Chem.268: 17651-17654 (1993)).

The ability to bind a substrate molecule with high affinity andspecificity is a prerequisite of a good enzyme. In addition, an enzymemust make use of well-positioned functional groups, either within itselfor a cofactor, to promote a particular chemical transformation.Furthermore, the enzyme must remain unchanged over the course of thereaction and be capable of operating with catalytic turnover. Some wouldadd the requirement that it be an informational macromolecule, comprisedof subunits whose specific ordering is responsible for catalyticactivity. While these criteria are open to debate on both semantic andchemical grounds, they serve to distinguish phenomena of chemical rateenhancement that range from simple solvent effects to biological enzymesoperating at the limit of substrate diffusion (Albery & Knowles,Biochemistry 15: 5631-5640 (1976)).

As described in greater detail hereinbelow, we sought to develop ageneral method for rapidly obtaining DNA catalysts and DNA enzymes,starting from random sequences. As an initial target, we chose areaction that we felt was well within the capability of DNA: thehydrolytic cleavage of an RNA phosphodiester, assisted by a divalentmetal cofactor. This is the same reaction that is carried out by avariety of naturally-occurring RNA enzymes, including the hammerhead andhairpin motifs. (See, e.g., Forster A. C. & Syrnons R. H., Cell 49:211-220 (1987); Uhlenbeck, Nature 328: 596-600 (1987); Hampel & Tritz,Biochemistry 28: 4929-4933 (1989)).

It has recently been shown that, beginning with a randomized library oftRNA molecules, one can obtain ribozymes that have Pb²⁺-dependent,site-specific RNA phosphoesterase activity at neutral pH (Pan &Uhlenbeck, Biochemistry 31: 3887-3895 (1992); Pan & Uhlenbeck, Nature358: 560-563 (1992)). This is analogous to the fortuitous self-cleavagereaction of yeast tRNA^(Phe) (Dirheimer & Werner, Biochimie 54: 127-144(1972)), which depends on specific coordination of a Pb²⁺ ion at adefined site within the tRNA. (See Rubin & Sundaralingam, J. Biomol.Struct. Dyn. 1: 639-646 (1983); Brown, et al., Biochemistry 24:4785-4801 (1985).)

As disclosed herein, our goals included the development of DNAs thatcould carry out Pb²⁺-dependent cleavage of a particular RNAphosphoester, initially presented within a short leader sequenceattached to the 5′ end of the DNA, and ultimately located within aseparate molecule that could be cleaved in an intermolecular fashionwith rapid catalytic turnover. These goals were successfully achieved,as described further below.

No assumptions were made as to how the DNA would interact with thetarget phosphoester and surrounding nucleotides. Beginning with a poolof approximately 10¹⁴ random 50mer sequences, in vitro selection wasallowed to run its course. After five rounds of selection carried outover four days, the population as a whole had attained the ability tocleave the target phosphoester in the presence of 1 mM Pb²⁺ at a rate ofabout 0.2 min⁻¹. This is an approximately 10⁵-fold increase compared tothe spontaneous rate of cleavage under the same reaction conditions.

Individuals were isolated from the population, sequenced, and assayedfor catalytic activity. Based on this information, the reaction wasconverted to an intermolecular format and then simplified to allowsite-specific cleavage of a 19mer substrate by a 38mer DNA enzyme, in areaction that proceeds with a turnover rate of 1 min⁻¹ at 23° C. and pH7.0 in the presence of 1 mM PbOAc.

B. In Vitro Selection Scheme

A starting pool of approximately 10¹⁴ single-stranded DNA molecules wasgenerated, all of which contain a 5′ biotin moiety, followedsuccessively by a fixed domain that includes a single ribonucleotide, apotential catalytic domain comprised of 50 random deoxyribonucleotides,and a second fixed domain that lay at the 3′ terminus (FIG. 1).

The pool was constructed by a nested PCR (polymerase chain reaction)technique, beginning with synthetic DNA that contained 50 randomnucleotides flanked by primer binding sites. The nested PCR primer was a5′-biotinylated synthetic oligodeoxynucleotide with a 3′-terminaladenosine ribonucleotide. Ribonucleotide-terminated oligonucleotidesefficiently prime template-directed elongation in the context of the PCR(L. E. Orgel, personal communication), in this case giving rise to anextension product that contains a single embedded ribonucleotide.

FIG. 1 illustrates a selective amplification scheme for isolation ofDNAs that cleave a target RNA phosphoester. Double-stranded DNAcontaining a stretch of 50 random nucleotides is amplified via PCR,employing a 5′-biotinylated DNA primer (e.g., primer 3—3a or 3b)terminated at the 3′ end by an adenosine ribonucleotide (represented bythe symbol “N” or “rA”, wherein both N and rA represent an adenosineribonucleotide). This primer is extended by Taq polymerase to yield aDNA product that contains a single embedded ribonucleotide. Theresulting double-stranded DNA is immobilized on a streptavidin matrixand the unbiotinylated DNA strand is removed by washing with 0.2 N NaOH.After re-equilibrating the column with a buffered solution, the columnis washed with the same solution with added 1 mM PbOAc. DNAs thatundergo Pb²⁺-dependent self-cleavage are released from the column,collected in the eluant, and amplified by PCR. The PCR products are thenused to initiate the next round of selective amplification.

The PCR products were passed over a streptavidin affinity matrix,resulting in noncovalent attachment of the 5′-biotinylated strand of theduplex DNA. The nonbiotinylated strand was removed by brief washing with0.2 N NaOH, and the bound strand was equilibrated in a buffer containing0.5 M NaCl, 0.5 M KCl, 50 mM MgCl₂, and 50 mM HEPES (pH 7.0) at 23° C.Next, 1 mM PbOAc was provided in the same buffer, allowingPb²⁺-dependent cleavage to occur at the target phosphoester, therebyreleasing a subset of the DNAs from the streptavidin matrix. Inprinciple, an individual DNA might facilitate its own release by variousmeans, such as disruption of the interaction between biotin andstreptavidin or cleavage of one of the deoxyribonucleotide linkages. Itwas felt that cleavage of the ribonucleoside 3′—O—P bond would be themost likely mechanism for release, based on the relative lability ofthis linkage, and that Pb²⁺-dependent hydrolytic cleavage would allowrelease to occur most rapidly. In principle, however, the in vitroselection procedure should identify the most favorable release mechanismas well as those individuals best able to carry out that mechanism.

DNA molecules released from the matrix upon addition of Pb²⁺ werecollected in the eluant, concentrated by precipitation with ethanol, andsubjected to nested PCR amplification. As in the construction of thestarting pool of molecules, the first PCR amplification utilized primersthat flank the random region (primers 1 and 2) and the second utilized a5′-biotinylated primer (primer 3b) that has a 3′-terminal riboadenylate,thereby reintroducing the target RNA phosphoester. The entire selectiveamplification procedure requires 3-4 hours to perform.

The molecules are purified in three ways during each round of thisprocedure: first, following PCR amplification, by extracting twice withphenol and once with chloroform/isoamyl alcohol, then precipitating withethanol; second, following attachment of the DNA to streptavidin, bywashing away all the nonbiotinylated molecules under strongly denaturingconditions; and third, following elution with Pb²⁺, by precipitatingwith ethanol. There is no gel electrophoresis purification step, andthus no selection pressure constraining the molecules to a particularlength.

C. Selection of Catalytic DNA

We carried out five successive rounds of in vitro selection,progressively decreasing the reaction time following addition of Pb²⁺ inorder to progressively increase the stringency of selection. Duringrounds 1 though 3, the reaction time was 1 hour; during round 4, thereaction time was 20 minutes; and during round 5, it was 1 minute. Thestarting pool of single-stranded DNAs, together with the population ofmolecules obtained after each round of selection, was assayed forself-cleavage activity under conditions identical to those employedduring in vitro selection (see FIG. 2).

For this assay, the molecules were prepared with a 5′ -³²P rather than a5′-biotin moiety, allowing detection of both the starting material andthe 5′ cleavage product. Following a 5-minute incubation, there was nodetectable activity in the initial pool (G0) or in the populationobtained after the first and second rounds of selection. DNAs obtainedafter the third round (G3) exhibited a modest level of activity; thisactivity increased steadily, reaching approximately 50% self-cleavagefor the DNAs obtained after the fifth round of selection (G5). Cleavagewas detected only at the target phosphoester, even after long incubationtimes. This activity was lost if Pb²⁺ was omitted from the reactionmixture.

FIG. 2 illustrates the self-cleavage activity of the starting pool ofDNA (G0) and populations obtained after the first through fifth roundsof selection (G1-G5). Reaction mixtures contained 50 mM MgCl₂, 0.5 MNaCl, 0.5 M KCl, 50 mM HEPES (pH 7.0 at 23° C.), and 3 nM [5′-³²P]-labeled DNA, incubated at 23° C. for 5 min either in the presenceor in the absence of 1 mM PbOAc. The symbol Pre represents108-nucleotide precursor DNA (SEQ ID NO 4); Clv, 28-nucleotide5′-cleavage product (SEQ ID NO 5); and M, primer 3a (SEQ ID NO 6),corresponding in length to the 5′-cleavage product.

The 28-nucleotide 5′ cleavage product (Clv) illustrated preferably hasthe sequence 5′-GGGACGAATTCTAATACGACTCACTATN-3′, wherein “N” representsadenosine ribonucleotide with an additional 2′,3′-cyclic phosphate onthe 3′ end (SEQ ID NO 5). In alternative embodiments, “N” representsadenosine ribonucleotide with an additional 2′ or 3′ phosphate on the 3′end of the molecule.

In FIG. 2, the “G0” lane “Pre” band comprises a sampling of108-nucleotide precursor DNAs that each include 50 random nucleotides.Therefore, any given “Pre” sampling will contain a wide variety ofprecursor DNAs, and each sampling will likely differ from previous andsubsequent samplings. The “G1” through “G5” lanes contain “Pre” bandsthat are increasingly enriched for catalytic DNA molecules, but stillcontain a large number of different DNA sequences (i.e., differing inthe 50 nucleotide randomized domain). A sample of these differentsequences from “G5 Pre” DNA is provided in FIG. 3.

Shotgun cloning techniques were employed to isolate individuals from theG5 population; the complete nucleotide sequences of 20 of thesesubclones were then determined (see FIG. 3). (Also see, e.g., Cadwelland Joyce, in PCR Methods and Applications 2: 28-33 (1992); Cadwell andJoyce, PCR Methods and Applications 3 (Suppl.): S136-S140 (1994).) Ofthe 20 sequences, five were unique, two occurred twice, one occurredthree times, and one occurred eight times. All of the individualvariants share common sequence elements within the 50-nucleotide regionthat had been randomized in the starting pool of DNA. They all containtwo presumed template regions, one with complementarity to a stretch ofnucleotides that lies just upstream from the cleavage site and the otherwith complementarity to nucleotides that lie at least four nucleotidesdownstream. Between these two presumed template regions lies a variabledomain of 1-11 nucleotides, followed by the fixed sequence 5′-AGCG-3′,then a second variable domain of 3-8 nucleotides, and finally the fixedsequence 5′-CG-3′ or 5′-CGA-3′. Nucleotides that lie outside of the twopresumed template regions are highly variable in both sequence andlength. In all of the sequenced subclones, the region corresponding tothe 50 initially-randomized nucleotides remains a total of 50nucleotides in length.

FIG. 3 illustrates the sequence alignment of individual variantsisolated from the population after five rounds of selection. The fixedsubstrate domain (5′-GGGACGAATTCTAATACGACTCACTATrAGGAAGAGATGGCGAC-3′, or5′-GGGACGAATTCTAATACGACTCACTATNGGAAGAGATGGCGAC-3′, where N representsadenosine ribonucleotide) (SEQ ID NO 13) is shown at the top, with thetarget riboadenylate identified with an inverted triangle. Substratenucleotides that are commonly involved in presumed base-pairinginteractions are indicated by a vertical bar. Sequences corresponding tothe 50 initially-randomized nucleotides are aligned antiparallel to thesubstrate domain. All of the variants are 3′-terminated by the fixedsequence 5′-CGGTAAGCTTGGCAC-3′ (SEQ ID NO 1) (“primer site”; not shown).Nucleotides within the initially-randomized region that are presumed toform base pairs with the substrate domain are indicated on the right andleft sides of the Figure; the putative base-pair-forming (or substratebinding) regions of the enzymatic DNA molecules are individually boxedin each sequence shown. The highly-conserved nucleotides within theputative catalytic domain are illustrated in the two boxed columns.

While it is anticipated that additional data will be helpful inconstructing a meaningful secondary structural model of the catalyticdomain, we note that, like the hammerhead and hairpin ribozymes, thecatalytic domain of our enzymatic DNA molecules appears to contain aconserved core flanked by two substrate binding regions (or recognitiondomains) that interact with the substrate through base-pairinginteractions. Similar to the hammerhead and hairpin ribozymes, thecatalytic DNAs also appear to require a short stretch of unpairedsubstrate nucleotides—in this case 5′-GGA-3′—between the two regionsthat are involved in base pairing.

It was also interesting to note that each of the nine distinct variantsexhibited a different pattern of presumed complementarity with thesubstrate domain. In some cases, base pairing was contiguous, while inothers it was interrupted by one or more noncomplementary pairs. Thegeneral tendency seems to be to form tighter interaction with thenucleotides that lie upstream from the cleavage site compared to thosethat lie downstream. Binding studies and site-directed mutagenesisanalysis should enable us to gain further insights and to furthersubstantiate this conjecture.

In order to gain further insight into the sequence requirements forcatalytic function, the self-cleavage activity of six of the ninevariants was tested and evaluated under the within-described selectionconditions (see FIG. 3). Not surprisingly, the sequence that occurred ineight of the 20 subclones proved to be the most reactive, with afirst-order rate constant of 1.4 min⁻¹. All of the studied variants wereactive in the self-cleavage assay and all gave rise to a single5′-labeled product corresponding to cleavage at the target RNAphosphoester.

The dominant subclone was further analyzed under a variety of reactionconditions. Its self-cleavage activity was dependent on Pb²⁺ but wasunaffected if Mg²⁺ was omitted from the reaction mixture. There was arequirement for a monovalent cation as well, which can be met by eitherNa⁺ or K⁺. The reaction rate increased linearly with increasingconcentration of monovalent cation over the range of 0-1.0 M (r=0.998).Other variables that may affect the reaction, such as pH, temperature,and the presence of other divalent metals, are in the process of beingevaluated further.

Example 2 Materials and Methods

A. Oligonucleotides and Oligonucleotide Analogs

Synthetic DNAs and DNA analogs were purchased from Operon Technologies.The 19-nucleotide substrate, 5′-pTCACTATrAGGAAGAGATGG-3′ (or5′-pTCACTATNGGAAGAGATGG-3′, wherein “N” represents adenosineribonucleotide) (SEQ ID NO 7), was prepared by reverse-transcriptasecatalyzed extension of 5′-pTCACTATrA-3′ (or 5′-pTCACTATN-3′, wherein “N”represents adenosine ribonucleotide) (SEQ ID NO 8), as previouslydescribed (Breaker, Banerji, & Joyce, Biochemistry 33: 11980-11986(1994)), using the template 5′-CCATCTCTTCCTATAGTGAGTCCGGCTGCA-3′ (SEQ IDNO 9). Primer 3,5′-GGGACGAATTCTAATACGACTCACTATrA-3′ (or5′-GGGACGAATTCTAATACGACTCACTATN-3′, wherein “N” represents adenosineribonucleotide) (SEQ ID NO 6), was either 5′-labeled with [y-³²P]ATP andT4 polynucleotide kinase (primer 3a) or 5′-thiophosphorylated with[y-S]ATP and T4 polynucleotide kinase and subsequently biotinylated withN-iodoacetyl-N′-biotinylhexylenediamine (primer 3b).

B. DNA Pool Preparation

The starting pool of DNA was prepared by PCR using the syntheticoligomer 5′-GTGCCAAGCTTACCG-N₅₀-GTCGCCATCTCTTCC-3′ (SEQ ID NO 4), whereN is an equimolar mixture of G, A, T and C. A 2-ml PCR, containing 500pmoles of the randomized oligomer, 1,000 pmoles primer 1(5′-GTGCCAAGCTTACCG-3′, SEQ ID NO 10), 500 pmoles primer 2(5′-CTGCAGAATTCTAATACGACTCACTATAGGAAGAGATGGCGAC-3′, SEQ ID NO 11), 500pmoles primer 3b, 10 μCi [α-³²P]dATP, and 0.2 U μl⁻¹ Taq DNA polymerase,was incubated in the presence of 50 mM KCl, 1.5 mM MgCl₂, 10 mM Tris-HCl(pH 8.3 at 23° C.), 0.01% gelatin, and 0.2 mM of each dNTP for 1 min at92° C., 1 min at 50° C., and 2 min at 72° C., then 5 cycles of 1 min at92° C., 1 min at 50° C., and 1 min at 72° C. The resulting mixture wasextracted twice with phenol and once with chloroform/isoamyl alcohol,and the DNA was isolated by precipitation with ethanol.

C. In Vitro Selection

The starting pool of DNA was resuspended in 500 μL of buffer A (1 M NaCland 50 mM HEPES (pH 7.0 at 23° C.)) and was passed repeatedly over astreptavidin column (AffiniTip Strep 20, Genosys, The Woodlands, Tex.).The column was washed with five 100-μl volumes of buffer A, followed byfive 100-μl volumes of 0.2 N NaOH, then equilibrated with five 100-μlvolumes of buffer B (0.5 M NaCl, 0.5 M KCl, 50 mM McCl₂, and 50 mM HEPES(pH 7.0 at 23° C.)). The immobilized single-stranded DNA was eluted overthe course cf 1 hr with three 20-μl volumes of buffer B with added 1 mMPbOAc. The entire immobilization and elution process was conducted at23° C. The eluant was collected in an equal volume of buffer C (50 mMHEPES (pH 7.0 at 23° C.) and 80 mM EDTA) and the DNA was precipitatedwith ethanol.

The resulting DNA was amplified in a 100-μL PCR containing 20 pmolesprimer 1, 20 pmoles primer 2, 0.05 U μl⁻¹ Taq polymerase, 50 mM KCl, 1.5mM MgCl₂, 10 mM Tris-HCl (pH 8.3 at 23° C.), 0.01% gelatin, and 0.2 mMof each dNTP for 30 cycles of 10 sec at 92° C., 30 sec at 50° C., and 30sec at 72° C. The reaction products were extracted twice with phenol andonce with chloroform/isoamyl alcohol, and the DNA was recovered byprecipitation with ethanol. Approximately 4 pmoles of the amplified DNAwas added to a second, nested PCR containing 100 pmoles primer 1, 100pmoles primer 3b, 20 μCi [α-³²P]dATP, and 0.1 U μl⁻¹ Taq polymerase, ina total volume of 200 μL that was amplified for 10 cycles of 1 min at92° C., 1 min at 50° C., and 1 min at 72° C. The PCR products were oncemore extracted and precipitated, and the resulting DNA was resuspendedin 50 μL buffer A, then used to begin the next round of selection.

The second and third rounds were carried out as above, except that thenested PCR at the end of the third round was performed in a 100-μlvolume. During the fourth round, the elution time following addition ofPb²⁺ was reduced to 20 min (two 20-μL elution volumes) and only half ofthe recovered DNA was used in the first PCR, which involved only 15temperature cycles. During the fifth round, the elution time was reducedto 1 min (two 20-μL elution volumes) and only one-fourth of therecovered DNA was used in the first PCR, which involved 15 temperaturecycles. DNA obtained after the fifth round of selection was subclonedand sequenced, as described previously (Tsang & Joyce, Biochemistry 33:5966-5973 (1994)).

D. Kinetic Analysis of Catalytic DNAs

Populations of DNA and various subcloned individuals were prepared witha 5′-³²P label by asymmetric PCR in a 25-μl reaction mixture containing10 pmoles primer 3a, 0.5 pmoles input DNA, and 0.1 U μl⁻¹ Taqpolymerase, under conditions as described above, for 10 cycles of 1 minat 92° C., 1 min at 50° C., and 1 min at 72° C. The resulting[5′-³²P]-labeled amplification products were purified by electrophoresisin a 10% polyacrylamide/8 M gel.

Self-cleavage assays were carried out following preincubation of the DNAin buffer B for 10 min. Reactions were initiated by addition of PbOAc to1 mM final concentration and were terminated by addition of an equalvolume of buffer C. Reaction products were separated by electrophoresisin a 10% polyacrylamide/8M gel. Kinetic assays under multiple-turnoverconditions were carried out in buffer B that included 50 μg ml⁻¹ BSA toprevent adherence of material to the vessel walls. Substrate and enzymemolecules were preincubated separately for 5 min in reaction buffer thatlacked Pb²⁺, then combined, and the reaction was initiated by additionof PbOAc to a final concentration of 1 mM.

Example 3 Evolution of Deoxyribozymes That Cleave Intermolecularly

A. Conversion to an Intermolecular Format

Based on the variable pattern of presumed base-pairing interactionsbetween the catalytic and substrate domains of the studied variants, itwas felt that it would be reasonably straightforward to convert theDNA-catalyzed reaction to an intermolecular format. In doing so, wewished to simplify the two substrate-binding regions of the catalyst sothat each would form an uninterrupted stretch of 7-8 base pairs with thesubstrate. In addition, we wished to provide a minimal substrate,limited to the two base-pairing regions and the intervening sequence5′-GGA-3′ (FIG. 4A).

FIGS. 4A and 4B illustrate DNA-catalyzed cleavage of an RNA phosphoesterin an intermolecular reaction that proceeds with catalytic turnover.FIG. 4A is a diagrammatic representation of the complex formed betweenthe 19mer substrate and 38mer DNA enzyme. The substrate contains asingle adenosine ribonucleotide (“rA” or “N”, adjacent to the arrow),flanked by deoxyribonucleotides. The synthetic DNA enzyme is a38-nucleotide portion of the most frequently occurring variant shown inFIG. 3. Highly-conserved nucleotides located within the putativecatalytic domain are “boxed”. As illustrated, one conserved sequence is“AGCG”, while another is “CG” (reading in the 5′→3′ direction).

FIG. 4B shows an Eadie-Hofstee plot used to determine K_(m) (negativeslope) and V_(max) (y-intercept) for DNA-catalyzed cleavage of[5′-³²P]-labeled substrate under conditions identical to those employedduring in vitro selection. Initial rates of cleavage were determined forreactions involving 5 nM DNA enzyme and either 0.125, 0.5, 1, 2, or 4 μMsubstrate.

In designing the catalytic domain, we relied heavily on the compositionof the most reactive variant, truncating by two nucleotides at the 5′end and 11 nucleotides at the 3′ end. The 15 nucleotides that laybetween the two template regions were left unchanged and a singlenucleotide was inserted into the 3′ template region to form a continuousstretch of nucleotides capable of forming base pairs with the substrate.The substrate was simplified to the sequence 5′-TCACTATrAGGAAGAGATGG-3′(or 5′-TCACTATNGGAAGAGATGG-3′, wherein “N” represents adenosineribonucleotide) (SEQ ID NO 12), where the underlined nucleotidescorrespond to the two regions involved in base pairing with thecatalytic DNA molecule.

The simplified reaction system, employing a 38mer catalytic DNA molecule(catalyst) comprised entirely of deoxyribonucleotides and a 19mersubstrate containing a single ribonucleotide embedded within anotherwise all-DNA sequence, allows efficient DNA-catalyzed phosphoestercleavage with rapid turnover. Over a 90-minute incubation in thepresence of 0.01 μM catalyst and 1 μM substrate, 46% of the substrate iscleaved, corresponding to 46 turnovers of the catalyst. A preliminarykinetic analysis of this reaction was carried out, evaluated undermultiple-turnover conditions. The DNA catalyst exhibits Michaelis-Mentenkinetics, with values for k_(cat) and K_(m) of 1 min and 2 μM,respectively (see FIG. 4B). The value for K_(m) is considerably greaterthan the expected dissociation constant between catalyst and substratebased on Watson-Crick interactions. The substrate was incubated underidentical reaction conditions (but in the absence of the catalyst); avalue for k_(uncat) of 4×10⁻⁶ min⁻¹ was obtained. This is consistentwith the reported value of 5×10⁻³ min⁻¹ for hydrolysis of the morelabile 1-nitrophenyl-1,2-propanediol in the presence of 0.5 mM Pb²⁺ atpH 7.0 and 37° C. (Breslow & Huang, PNAS USA 88: 4080-4083 (1991)).

It is now presumed that the phosphoester cleavage reaction proceeds viaa hydrolytic mechanism involving attack by the ribonucleoside2′-hydroxyl on the vicinal phosphate, generating a 5′ product with aterminal 2′(3′)-cyclic phosphate and 3′ product with a terminal5′-hydroxyl. In support of this mechanism, the 3′-cleavage product isefficiently pliosphorylated with T4 polynucleotide kinase and[γ-³²P]ATP, consistent with the availability of a free 5′-hydroxyl (datanot shown).

B. Discussion

After five rounds of in vitro selection, a population of single-strandedDNA molecules that catalyze efficient Pb²⁺-dependent cleavage of atarget RNA phosphoester was obtained. Based on the common features ofrepresentative individuals isolated from this population, a simplifiedversion of both the catalytic and substrate domains was constructed,leading to a demonstration of rapid catalytic turnover in anintermolecular context. Thus the 38mer catalytic domain provides anexample of a DNA enzyme, or what might be termed a “deoxyribozyme”.

Referring to this molecule as an enzyme, based on the fact that it is aninformational macromolecule capable of accelerating a chemicaltransformation in a reaction that proceeds with rapid turnover and obeysMichaelis-Menten kinetics, may not satisfy everyone's notion of whatconstitutes an enzyme. Some might insist that an enzyme, by definition,must be a polypeptide. If, however, one accepts the notion of an RNAenzyme, then it seems reasonable to adopt a similar view concerning DNAenzymes. Considering how quickly we were able to generate this moleculefrom a pool of random-sequence DNAs, we expect that many other examplesof synthetic DNA enzymes will appear in the near future.

The Pb²⁺-dependent cleavage of an RNA phosphoester was chosen as aninitial target for DNA catalysis because it is a straightforwardreaction that simply requires the proper positioning of a coordinatedPb²⁺-hydroxyl to facilitate deprotonation of the 2′ hydroxyl that liesadjacent to the cleavage site. (See, e.g., Pan, et al., in The RNAWorld, Gesteland & Atkins (eds.), pp. 271-302, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1993).) Pb²⁺ is known tocoordinate to the N7 position of purines, the O6 position of guanine,the O4 position of uracil, and the N3 position of cytosine (Brown, etal., Nature 303: 543-546 (1993)). Thus, the differences in sugarcomposition and conformation of DNA compared to RNA seemed unlikely toprevent DNA from forming a well-defined Pb²⁺-binding pocket.

A substrate that contains a single ribonucleotide within an otherwiseall-DNA sequence was chosen because it provided a uniquely favored sitefor cleavage and insured that any resulting catalytic activity would beattributable solely to DNA. Substrate recognition appears to depend ontwo regions of base-pairing interactions between the catalyst andsubstrate. However, the unpaired substrate nucleotides, 5′-GGA-3′, thatlie between these two regions may play an important role in substraterecognition, metal coordination, or other aspects of catalytic function.

It is further anticipated that an all-RNA molecule, other RNA-DNAcomposites, and molecules containing one or more nucleotide analogs maybe acceptable substrates. As disclosed herein, the within-described invitro evolution procedures may successfully be used to generateenzymatic DNA molecules having the desired specificities; furtheranalyses along these lines are presently underway.

In addition, studies to determine whether the presumed base-pairinginteractions between enzyme and substrate are generalizable with respectto sequence are in progress, using the presently-described methods. Thewithin-disclosed Pb²⁺-dependent deoxyribozymes may also be consideredmodel compounds for exploring the structural and enzymatic properties ofDNA.

The methods employed in the present disclosure for the rapid developmentof DNA catalysts will have considerable generality, allowing us toutilize other cofactors to trigger the cleavage of a target linkageattached to a potential catalytic domain. In this regard, thedevelopment of Mg²⁺-dependent DNA enzymes that specifically cleavetarget RNAs under physiological conditions is of interest, as is thedevelopment of DNA enzymes that function in the presence of othercations (see Example 4). Such molecules will provide an alternative totraditional antisense and ribozyme approaches for the specificinactivation of target mRNAs.

DNA thus joins RNA and protein on the list of biological macromoleculesthat are capable of exhibiting enzymatic activity. The full extent ofDNA's catalytic abilities remains to be explored, but these explorationsshould proceed rapidly based on in vitro selection methods such as thoseemployed in this study.

DNA enzymes offer several important advantages compared to othermacromolecular catalysis. First, they are easy to prepare, in an erawhen most laboratories have access to an automated DNA synthesizer andthe cost of DNA phosphoramidites has become quite modest. Second, theyare very stable compounds, especially compared to RNA, thus facilitatingtheir use in biophysical studies. Third, we expect that they can beadapted to therapeutic applications that at present make use ofantisense DNAs that lack RNA-cleavage activity. In vitro selection couldbe carried out with DNA analogs, including compounds that are nucleaseresistant such as phosphorothioate-containing DNA, so long as theseanalogs can be prepared in the form of a deoxynucleoside 5′-triphosphateand are accepted as a substrate by a DNA-dependent DNA polymerase.Finally, DNA enzymes offer a new window on our understanding of themacromolecular basis of catalytic function. It will be interesting, forexample, to carry out comparative analyses of protein-, RNA-, andDNA-based enzymes that catalyze the same chemical transformation.

Example 4 Other Families of Catalytic DNAs

A starting pool of DNA was prepared by PCR essentially as described inExample 2.B. above, except that the starting pool of DNA comprisedmolecules containing 40 random nucleotides. Thus, the starting pool ofDNA described herein was prepared by PCR using the synthetic oligomer 5′GGG ACG AAT TCT AAT ACG ACT CAC TAT rA 25 GG AAG AGA TGG CGA CAT CTCN₄₀GT GAC GGT AAG CTT GGC AC 3′ (SEQ ID NO 23), where N is an equimolarmixture of G, A, T and C, and where the DNA molecules were selected forthe ability to cleave the phosphoester following the target rA. (SeeFIG. 6A, also.)

Selective amplification was carried out in the presence of eitherPb²⁺,Zn2+,Mn²⁺, or Mg²⁺, thereby generating at least four “families” ofcatalytic DNA molecules. As illustrated in FIG. 5, catalytic DNAmolecules demonstrating specific activity were generated in the presenceof a variety of cations.

FIG. 5 is a photographic representation showing a polyacrylamide geldemonstrating specific endoribonuclease activity of four families ofselected catalytic DNAs. Selection of a Pb²⁺-dependent family ofmolecules was repeated in a side-by-side fashion as a control. In eachgroup of three lanes, the first lane shows the lack of activity of theselected population in the absence of the metal cation, the second laneshows the observed activity in the presence of the metal cation, and thethird lane shows the lack of activity of the starting pool (G0). Atpresent, the order of reactivity is 1625 observed to bePb²⁺>Zn²⁺>Mn²⁺>Mg²⁺, mirroring the PK_(a) of the correspondingmetal-hydroxide.

After either five (G5) or six (G6) rounds of selective amplification inthe presence of the preselected divalent cation, the desiredendonuclease activity was obtained. The following description ofselective amplification in the presence of Mg²⁺ is intended to beexemplary.

Six rounds of in vitro selective amplification were carried out,following the method described in Example 2 hereinabove, except that thedivalent metal used was 1 mM Mg²⁺ rather than 1 mM Pb²⁺. (See alsoBreaker and Joyce, Chem. & Biol. 1: 223-229 (1994), incorporated byreference herein, which describes essentially the same procedure.)

Individual clones were isolated following the sixth round, and thenucleotide sequence of 24 of these clones was determined. All of thesequences began with: 5′ GGG ACG AAT TCT AAT ACG ACT CAC TAT rA GG AAGAGA TGG CGA CA (SEQ ID NO 23 from position 1 to 44) and ended with: CGGTAA GCT TGG CAC 3′ (SEQ ID NO 23 from position 9:3 to 107).

The segment in the middle, corresponding to TCTC N₄₀ GTGA (SEQ ID NO 23from position 45 to 92) in the starting pool, varied as follows:

CCG CCC ACC TCT TTT ACG AGC CTG TAC GAA ATA GTG CTC TTG TTA GTA T (SEQID NO 24)  (13)

TCT CTT CAG CGA TGC ACG CTT GTT TTA ATG TTG CAC CCA TGT TAG TGA (SEQ IDNO 25)  (5)

TCT CAT CAG CGA TTG AAC CAC TTG GTG GAC AGA CCC ATG TTA GTG A (SEQ ID NO26)  (2)

CCG CCC ACC TCT TTT ACG AGC CTG TAC GAA ATA GTG TTC TTG TTA GTA T (SEQID NO 27)  (1)

CCG CCC ACC TCT TTT ACG AGC CTG TAC GAA ATA GTG CTC TCG TTA GTA T (SEQID NO 28)  (1)

TCT CAG ACT TAG TCC ATC ACA CTC TGT GCA TAT GCC TGC TTG ATG TGA (SEQ IDNO 29)  (1)

-CT CTC ATC TGC TAG CAC GCT CGA ATA GTG TCA GTC GAT GTG A (SEQ ID NO30).  (1)

The initial number in parentheses indicates the number of clones havingthat particular sequence. Note that some mutations (highlighted in boldtype) occurred at nucleotide positions other than those that wererandomized initially.

The second sequence listed above (i.e., SEQ ID NO 25), which occurred in5 of 24 clones, was chosen as a lead (i.e. principal) compound forfurther study. Its cleavage activity was measured in the presence of a 1mM concentration of various divalent metals and 1 M NaCl at pH 7.0 and23° C.:

metal k_(obs) (min⁻¹) none n.d. Mg²⁺ 2.3 × 10⁻³ Mn²⁺ 6.8 × 10⁻³ Zn²⁺ 4.2× 10⁻² Pb²⁺ 1.1 × 10⁻²

Thus, the lead compound is active in the presence of all four divalentmetals, even though it was selected for activity in the presence ofMg²⁺. Conversely, DNA molecules that were selected for activity in thepresence of Mn²⁺, Zn²⁺, or Pb²⁺ did not show any activity in thepresence of Mg²⁺.

In addition, the population of DNAs obtained after six rounds of invitro selection in the presence of Mg²⁺, when prepared asall-phosphorothioate-containing DNA analogs, showed Mg²⁺-dependentcleavage activity at an observed rate of ˜10⁻³ min⁻¹. Thephosphorothioate-containing analogs were prepared enzymatically so as tohave an R_(P) configuration at each stereocenter. Such compounds arerelatively resistant to degradation by cellular nucleases compared tounmodified DNA.

The lead compound was re-randomized at 40 nucleotide positions(underlined), introducing mutations at a frequency of 15% (5%probability of each of the three possible base substitutions). There-randomized population was subjected to seven additional rounds of invitro selection. During the last four rounds, molecules that werereactive in the presence of 1 mM Pb²⁺ were removed from the populationbefore the remainder were challenged to react in the presence of 1 mMMg²⁺. Individual clones were isolated following the seventh round andthe nucleotide sequence of 14 of these clones was determined. All of thesequences began with: 5′ GGG ACG AAT TCT AAT ACG ACT CAC TAT rA GG AAGAGA TGG CGA CAT CTC (SEQ ID NO 23, from position 1 to 48), and endedwith: GTG ACG GTA AGC TTG GCA C 3′ (SEQ ID NO 23, from position 89 to107).

The segment in the middle, corresponding to the 40 partially-randomizedpositions (N₄₀, SEQ ID NO 23, from position 49 to 88), varied asfollows:

TAC AGC GAT TCA CCC TTG TTT AAG GGT TAC ACC CAT GTT A (SEQ ID NO31)  (4)

ATC AGC GAT TAA CGC TTG TTT CAA TGT TAC ACC CAT GTT A (SEQ ID NO32)  (2)

TTC AGC GAT TAA CGC TTA TTT TAG CGT TAC ACC CAT GTT A (SEQ ID NO33)  (2)

ATC AGC GAT TCA CCC TTG TTT TAA GGT TGC ACC CAT GTT A (SEQ ID NO34)  (1)

ATC AGC GAT TCA CCC TTG TTT AAG CGT TAC ACC CAT GTT G (SEQ ID NO35)  (1)

ATC AGC GAT TCA CCC TTG TTT TAA GGT TAC ACC CAT GTT A (SEQ ID NO36)  (1)

ATC AGC GAT TAA CGC TTA TTT TAG CGT TAC ACC CAT GTT A (SEQ ID NO37)  (1)

ATC AGC GAT TAA CGC TTG TTT TAG TGT TGC ACC CAT GTT A (SEQ ID NO38)  (1)

ATC AGC GAT TAA CGC TTA TTT TAG CAT TAC ACC CAT GTT A (SEQ ID NO39).  (1)

The number in parentheses indicates the number of clones having thatparticular sequence. Nucleotides shown in bold are those that differcompared to the lead compound.

Formal analysis of the cleavage activity of these clones is ongoing. Thepopulation as a whole exhibits Mg²⁺-dependent cleavage activity at anobserved rate of ˜10⁻² min⁻¹, with a comparable level of activity in thepresence of Pb²⁺.

FIGS. 6A and 6B provide two-dimensional illustrations of a “progenitor”catalytic DNA molecule and one of several catalytic DNA moleculesobtained via the selective amplification methods disclosed herein,respectively. FIG. 6A illustrates an exemplary molecule from thestarting pool, showing the overall configuration of the moleculesrepresented by SEQ ID NO 23. As illustrated, various complementarynucleotides flank the random (N₄₀) region.

FIG. 6B is a diagrammatic representation of one of the Mg²⁺-dependentcatalytic DNA molecules (or “DNAzymes”) generated via thewithin-described procedures. The location of the ribonucleotide in thesubstrate nucleic acid is indicated via the arrow. (The illustratedmolecule includes the sequence identified herein as SEQ ID NO 25, aswell as “beginning” and “ending” sequences of SEQ ID NO 23.)

Endonuclease activity is continuing to be enhanced in each of theaforementioned “families” via in vitro evolution, as disclosed herein,so it is anticipated that enzymatic DNA molecules of increasinglydesirable specificities may be generated successfully using thewithin-disclosed guidelines.

Example 5 Cleavage of Larger RNA Sequences

As an extension of the foregoing, we have developed DNA enzymes thatcleave an all-RNA substrate, rather than a single ribonucleotideembedded within an otherwise all-DNA substrate as demonstrated above.(Also see R. R. Breaker & G. F. Joyce, Chem. & Biol. 1: 223-229 (1994);R. R. Breaker & G. F. Joyce, Chem. & Biol. 2: 655-660 (1995)). As atarget sequence, we chose a stretch of 12 highly-conserved nucleotideswithin the U5 LTR region of HIV-1 RNA, having the sequence

5′ GUAACUAGAGAU 3′  (SEQ ID NO 49).

Following the methods described in the previous examples, we generated apool of 1014 DNA molecules that have the following composition:

5′- GGAAAA r(GUAACUAGAGAU) GGAAGAGATGGCGAC N₅₀ CGGTAAGCTTGGCAC -3′  (SEQID NO 50),

where N is an equimolar mixture of the deoxyribonucleotides G, A, T, andC, and where the sequence identified as “r(GUAACUAGAGAU)” is comprisedof ribonucleotides. (Optionally, one may alter the initial 5′ nucleotidesequence, e.g., by adding an additional dA residue to the sequencepreceding the ribonucleotide portion at the 5′ end, thus causing theinitial sequence to read “GGAAAAA” and causing SEQ ID NO 50 to be 99residues in length. Clearly, this is but one example of themodifications that may be made in order to engineer specific enzymaticDNA molecules, as disclosed in detail herein.)

The enzymatic DNA molecules thus produced were selected for theirability to cleave a phosphoester that lies within the embedded RNAtarget sequence. Ten rounds of in vitro selective amplification werecarried out, based on the enzymatic DNA 30 molecules' activity in thepresence of 10 mM Mg²⁺ at pH 7.5 and 37° C. During the selectionprocess, there was competition for “preferred” cleavage sites as well asfor the “best” catalyst that cleaves at each such preferred site. Twosites and two families of catalysts emerged as possessing the mostefficient cleavage capabilities (see FIG. 7).

FIG. 7 illustrates some of the results of ten rounds of in vitroselective amplification carried out essentially as described herein. Asshown, two sites and two families of catalysts emerged as displaying themost efficient cleavage of the target sequence. Cleavage conditions wereessentially as indicated in FIG. 7, namely, 10 mM Mg²⁺, pH 7.5, and 37°;data collected after the reaction ran for 2 hours is shown. Cleavage (%)is shown plotted against the number of generations (here, 0 through 10).The number/prevalence of catalytic DNA molecules capable of cleaving thetarget sequence at the indicated sites in the substrate is illustratedvia the vertical bars, with cleavage at G↓UAACUAGAGAU (SEQ ID NO 49)shown by the striped bars, and with cleavage at GUAACUA↓GAGAU (SEQ ID NO49) illustrated via the open (lightly-shaded) bars. In FIG. 7, asherein, the arrow (↓) indicates the site between two neighboringnucleotides at which cleavage occurs.

Various individuals from the population obtained after the 8th and 10throunds of selective amplification were cloned. The nucleotide sequencesof 29 individuals from the 8th round and 32 individuals from the 10thround were then determined (see Tables 2 and 3, respectively).

Under the heading “Nucleotide Sequence” in each of Tables 2 and 3 isshown the portion of each identified clone that corresponds to the 50nucleotides that were randomized in the starting pool (i.e., N₅₀); thus,the entire nucleotide sequence of a given clone generally includes thenucleotide sequences preceding, following, and including the “N₅₀”segment, presuming the substrate sequence is attached and thatself-cleavage has not occurred. For example, the entire sequence of a(non-self-cleaved) clone may generally comprise residue nos. 1-33 of SEQID NO 50, followed by the residues representing the randomized N₅₀region, followed by residue nos. 84-98 of SEQ ID NO 50, or by residuenos. 1-34 of SEQ ID NO 51, followed by the residues representing therandomized N₅₀ region, followed by residue nos. 85-99 of SEQ ID NO 51.It is believed, however, that the N₅₀ (or N₄₀) region—or a portionthereof—of each clone is particularly important in determining thespecificity and/or activity of a particular enzymatic DNA molecule. Thisis particularly evident in reactions in which the substrate and theDNAzyme are separate molecules (see, e.g., FIGS. 8 and 9).

Clone numbers are designated as 8-x or 10-x for individuals obtainedafter the 8th or 10th rounds, respectively. SEQ ID NOS are also listedand correspond to the “N₅₀” region of each clone.

TABLE 2 Cloned Individuals from 8th Round of Amplification Clone SEQ No.ID NO “N₅₀” Nucleotide Sequence (5′→3′) 8-2 52 CCA ATA GTG CTA CTG TGTATC TCA ATG CTG GAA ACA CGG GTT ATC TCC CG 8-4 53 CCA AAA CAG TGG AGCATT ATA TCT ACT CCA CAA AGA CCA CTT TTC TCC CG 8-5¹ 54 ATC CGT ACT AGCATG CAG ACA GTC TGT CTG CTT TTT CAT TAC TCA CTC CC 8-14 55 CAA TTC ATGATG ACC AAC TCT GTC AAC ACG CGA ACT TTT AAC ACT GGC A 8-17² 56 CTT CCACCT TCC GAG CCG GAC GAA GTT ACT TTT TAT CAC ACT ACG TAT TG 8-3 57 GGCAAG AGA TGG CAT ATA TTC AGG TAA CTG TGG AGA TAC CCT GTC TGC CA 8-6 58CTA GAC CAT TCA CGT TTA CCA AGC TAT GGT AAG AAC TAG AAT CAC GCG TA 8-859 CGT ACA CGT GGA AAA GCT ATA AGT CAA GTT CTC ATC ATG TAC CTG ACC GC8-10 60 CAG TGA TAC ATG AGT GCA CCG CTA CGA CTA AGT CTG TAA CTT ATT CTACC 8-22 61 ACC GAA TTA AAC TAC CGA ATA GTG TGG TTT CTA TGC TTC TTC TTCCCT GA 8-11 62 CAG GTA GAT ATA ATG CGT CAC CGT GCT TAC ACT CGT TTT ATTAGT ATG TC 8-21 63 CCC TAC AAC ACC ACT GGG CCC AAT TAG ATT AAC GCT ATTTTA TAA CTC G 8-12 64 CCA AAC GGT TAT AAG ACT GAA AAC TCA ATC AAT AGCCCA ATC CTC GCC C 8-13 65 CAC ATG TAT ACC TAA GAA ATT GGT CCC GTA GACGTC ACA GAC TTA CGC CA 8-23 66 CAC AAC GAA AAC AAT CTT CCT TGG CAT ACTGGG GAG AAA GTC TGT TGT CC 8-40 67 CAC ACG AAC ATG TCC ATT AAA TGG CATTCC GTT TTT CGT TCT ACA TAT GC 8-24 68 CAG AAC GAG GGT CTT GTA AGA CTACAC CTC CTC AGT GAC AAT AAT CCT G 8-26 69 CAC TAC AGC CTG ATA TAT ATGAAG AAC AGG CAA CAA GCT TAT GCA CTG G 8-27 70 GGG TAC ATT TAT GAT TCTCTT ATA AAG AGA ATA TCG TAC TCT TTT CCC CA 8-28 71 CCA AAG TAC ATT CCAACC CCT TAT ACG TGA AAC TTC CAG TAG TTT CCT A 8-29 72 CTT GAA GAT CCTCAT AAG ACG ATT AAA CAA TCC ACT GGA TAT AAT CCG GA 8-34 73 CGA ATA GTGTCC ATG ATT ACA CCA ATA ACT GCC TGC CTA TCA TGT TTA TG 8-35 74 CCA AGAGAG TAT CGG ATA CAC TTG GAA CAT AGC TAA CTC GAA CTG TAC CA 8-36 75 CCACTG ATA AAT AGG TAA CTG TCT CAT ATC TGC CAA TCA TAT GCC GTA 8-37 76 CCCAAA TTA TAA ACA ATT TAA CAC AAG CAA AAG GAG GTT CAT TGC TCC GC 8-39 77CAA TAA ACT GGT GCT AAA CCT AAT ACC TTG TAT CCA AGT TAT CCT CCC CC¹identical to 10-4, 10-40 ²identical to 8-20, 8-32, 8-38, 10-1, 10-34; 1mutation to 10-11; 3 mutations to 10-29

TABLE 3 Cloned Individuals from l0th Round of Amplification Clone SEQNo. ID NO “N₅₀” Nucleotide Sequence (5′→3′) 10-3³ 78 CCG AAT GAC ATC CGTAGT GGA ACC TTG CTT TTG ACA CTA AGA AGC TAC AC 10-10 79 CCA TAA CAA ATACCA TAG TAA AGA TCT GCA TTA TAT TAT ATC GGT CCA CC 1O-12 80 CAG AAC AAAGAT CAG TAG CTA AAC ATA TGG TAC AAA CAT ACC ATC TCG CA 10-14 81 CCT TTAGTT AGG CTA GCT ACA ACG ATT TTT CCC TGC TTG GCA ACG ACA C 10-15 82 CTCCCT ACG TTA CAC CAG CGG TAC GAA TTT TCC ACG AGA GGT AAT CCG CA 10-19 83CGG CAC CTC TAG TTA GAC ACT CCG GAA TTT TTC CCC 10-39 84 CGG CAC CTC TAGTTA GAC ACT CCG GAA TTT TAG CCT ACC ATA GTC CGG T 10-23 85 CCC TTT GGTTAG GCT AGC TAC AAC GAT TTT TCC CTG CTT GAA TTG TA 10-27⁴ 86 CCC TTT GGTTAG GCT AGC TAC AAC GAT TTT TCC CTG CTT GAC CTG TTA CGA 10-31 87 CCT TTAGTT AGG CTA GCT ACA ACG ATT TTT CCC TGC TTG GAA CGA CAC 10-18 88 CAT GGCTTA ATC ATC CTC AAT AGA AGA CTA CAA GTC GAA TAT GTC CCC CC 10-20 89 CAACAG AGC GAG TAT CAC CCC CTG TCA ATA GTC GTA TGA AAC ATT GGG CC 10-6 90TAC CGA CAA GGG GAA TTA AAA GCT AGC TGG TTA TGC AAC CCT TTT CGC A 10-791 CTC GAA ACA GTG ATA TTC TGA ACA AAC GGG TAC TAC GTG TTC AGC CCC C10-8 92 CCA ATA ACG TAA CCC GGT TAG ATA AGC ACT TAG CTA AGA TGT TTA TCCTG 10-16 93 CAA TAC AAT CGG TAC GAA TCC AGA AAC ATA ACG TTG TTT CAG AATGGT CC 10-21 94 GCA ACA ACA AGA ACC AAG TTA CAT ACA CGT TCA TCT ATA CTGAAC CCC CA 10-24 95 CCT TTG AGT TCC TAA ATG CCG CAC GGT AAG CTT GGC ACACTT TGA CTG TA 10-28 96 CAA AGA TCT CAC TTT GGA AAT GCG AAA TAT GTA TATTCG CCC TGT CTG C 10-33 97 CCA CGT AGA ATT ATC TGA TTT ATA ACA TAA CGCAGG ATA ACT CTC GCC CA 10-35 98 CAC AAG AAA GTG TCG TCT CCA GAT ATT TGAGTA CAA GGA ACT ACG CCC 10-36 99 CAT GAA GAA ATA GGA CAT TCT ACA GGC TGGACC GTT ACT ATG CCT GTA GG 10-37 100 CAT AGG ATA ATC ATG GCG ATG CTT ATGACG TGT ACA TCT ATA CCT T 10-38 101 CAG ATG ATC TTC CTT TAA AGA CTA CCCTTT AAA GAA ACA TAA GGT ACC CC ³1 mutation to 10-5 ⁴1 mutation to 10-30

The self-cleavage activity of various clones was subsequently measured.Clones 8-5, 8-17, and 10-3 were found to cleave efficiently at the site5′ GUAACU↓AGAGAU (SEQ ID NO 49) 3′, while clones 10-14, 10-19 and 10-27were found to cleave efficiently at the site 5′ G↓UAACUAGAGAU 3′ (SEQ IDNO 49). When the RNA portion of the molecule was extended to thesequence 5′ GGAAAAAGUAACUAGAGAUGGAAG 3′ (residue nos. 1-24 of SEQ ID NO51), clones 8-17, 10-14, and 10-27 retained full activity, while clones8-5, 10-3, and 10-19 showed diminished activity. Subsequently, clone10-23 was found to exhibit a high level of activity in the self-cleavagereaction involving the extended RNA domain.

It should also be noted, in the event one of skill in the relevant artdoes not appreciate same, that the nucleotide sequences preceding andfollowing the “N₅₀” segments of the polynucleotide molecules engineeredaccording to the teachings of the present invention disclosure may bealtered in a variety of ways in order to generate enzymatic DNAmolecules of particular specificities. For example, while residue nos.1-24 of SEQ ID NO 51 are described herein as RNA nucleotides, they mayalternatively comprise DNA, RNA, or composites thereof. (Thus, forexample, SEQ ID NO 51 could easily be altered so that nucleic acidresidue nos. 1-7 would comprise DNA, residue nos. 8-19 would compriseRNA, residue nos. 20-99 would comprise DNA, and so on.) Similarly, thenucleotides following the “N₅₀” region may comprise RNA, DNA, orcomposites thereof. The length of the regions preceding and followingthe “N₅₀” (or “N₄₀”—see Example 4) region(s) may also be varied, asdisclosed herein. Further, sequences preceding and/or following N₅₀ orN₄₀ regions may be shortened, expanded, or deleted in their entirely.

Moreover, as noted above, we selected a specific region of HIV-1 RNA asthe target sequence in the methods described in this Example; such asequence is not the only sequence one may use as a target. Clearly, oneof skill in the relevant art may follow our teachings herein to engineerand design enzymatic DNA molecules with specificity for other targetsequences. As disclosed herein, such target sequences may be constructedor inserted into larger sequences comprising DNA, RNA, or compositesthereof, as illustrated by SEQ ID NOS 50 and 51.

The self-cleavage reaction was easily converted to an intermolecularcleavage reaction by dividing the enzyme and substrate domains intoseparate molecules. Clones 8-17 and 10-23 were chosen as prototypemolecules. Both were shown to act as DNA enzymes in the cleavage of aseparate all-RNA substrate in a reaction that proceeds with multipleturnover (FIG. 8). The substrate binding arms were subsequently reducedto 7 base-pairs on each side of the unpaired nucleotide that demarcatesthe cleavage site (FIG. 9).

FIG. 8 illustrates the nucleotide sequences, cleavage sites, andturnover rates of two catalytic DNA molecules of the present invention,clones 8-17 and 10-23. Reaction conditions were as shown, namely, 10 mMMg²⁺, pH 7.5, and 37° C. The DNAzyme identified as clone 8-17 isillustrated on the left, with the site of cleavage of the RNA substrateindicated by the arrow. The substrate sequence(5′-GGAAAAAGUAACUAGAGAUGGAAG - 3′) (residue nos. 1-24 of SEQ ID NO51)—which is separate from the DNAzyme (i.e., intermolecular cleavage isshown)—is labeled as such. Similarly, the DNAzyme identified herein as10-23 is shown on the right, with the site of cleavage of the RNAsubstrate indicated by the arrow. Again, the substrate sequence isindicated. For the 8-17 enzyme, the turnover rate was approximately 0.6hr⁻¹; for the 10-23 enzyme, the turnover rate was approximately 1 hr⁻¹.

As illustrated in FIG. 8, the nucleotide sequence of the clone 8-17catalytic DNA molecule capable of cleaving a separate substrate moleculewas as follows: 5′-CTTCCACCTTCCGAGCCGGACGAAGTTACTTTTT-3′ (residue nos.1-34 of SEQ ID NO 56). In that same figure, the nucleotide sequence ofthe clone 10-23 catalytic DNA molecule capable of cleaving a separatesubstrate molecule was as follows: 5′-CTTTGGTTAGGCTAGCTACAACGATTTTTCC-3′(residue nos. 3-33 of SEQ ID NO 85).

FIG. 9 further illustrates the nucleotide sequences, cleavage sites, andturnover rates of two catalytic DNA molecules of the present invention,clones 8-17 and 10-23. Reaction conditions were as shown, namely, 10 mMMg²⁺, pH 7.5, and 37° C. As in FIG. 8, the DNAzyme identified as clone8-17 is illustrated on the left, with the site of cleavage of the RNAsubstrate indicated by the arrow. The substrate sequence(5′-GGAAAAAGUAACUAGAGAUGGAAG - 3′) (residue nos. 1-24 of SEQ ID NO51)—which is separate from the DNAzyme (i.e., intermolecular cleavage isshown)—is labeled as such. Similarly, the DNAzyme identified herein as10-23 is shown on the right, with the site of cleavage of the RNAsubstrate indicated by the arrow. Again, the substrate sequence isindicated. For the 8-17 enzyme, k_(obs) was approximately 0.002 min⁻¹;for the 10-23 enzyme, the value of k_(obs) was approximately 0.01 min⁻¹.

As illustrated in FIG. 9, the nucleotide sequence of the clone 8-17catalytic DNA molecule capable of cleaving a separate substrate moleculewas as follows: 5′-CCACCTTCCGAGCCGGACGAAGTTACT-3′ (residue nos. 4-30 ofSEQ ID NO 56). In that same figure, the nucleotide sequence of the clone10-23 catalytic DNA molecule capable of cleaving a separate substratemolecule was as follows: 5′-CTAGTTAGGCTAGCTACAACGATTTTTCC-3′ (residuenos. 5-33 of SEQ ID NO 85, with “CTA” substituted for “TTG” at the 5′end).

The catalytic rate of the RNA-cleaving DNA enzymes has yet to be fullyoptimized. As disclosed above and as reported in previous studies, wehave been able to improve the catalytic rate by partially randomizingthe prototype molecule and carrying out additional rounds of selectiveamplification. We have found, however, that the K_(m) for Mg²⁺ isapproximately 5 mM and 2 mM for the 8-17 and 10-23 DNA enzymes,respectively, measured at pH 7.5 and 37° C.; this is certainlycompatible with intracellular conditions.

The foregoing specification, including the specific embodiments andexamples, is intended to be illustrative of the present invention and isnot to be taken as limiting. Numerous other variations and modificationscan be effected without departing from the true spirit and scope of thepresent invention.

101 1 15 DNA Artificial Sequence Description of Artificial Sequence 3′terminal sequence 1 cggtaagctt ggcac 15 2 20 DNA Artificial SequenceDescription of Combined DNA/RNA Molecule The N at position 8 isadenosine ribonucleotide. 2 tcactatnag gaagagatgg 20 3 38 DNA ArtificialSequence Description of Artificial Sequence DNA enzyme 3 acacatctctgaagtagcgc cgccgtatag tgacgcta 38 4 80 DNA Artificial SequenceDescription of Artificial Sequence oligomer 4 gtgccaagct taccgnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnngtcgc catctcttcc 805 28 DNA Artificial Sequence Description of Combined DNA/RNA MoleculeThe n at position 28 is adenosine ribonucleotide. 5 gggacgaattctaatacgac tcactatn 28 6 28 DNA Artificial Sequence Description ofCombined DNA/RNA Molecule The n at position 28 is adenosineribonucleotide. 6 gggacgaatt ctaatacgac tcactatn 28 7 19 DNA ArtificialSequence Description of Combined DNA/RNA Molecule The n at position 8 isadenosine ribonucleotide. 7 tcactatngg aagagatgg 19 8 8 DNA ArtificialSequence misc_feature (8) The n at position 8 is adenosine nucleotide. 8tcactatn 8 9 30 DNA Artificial Sequence Description of ArtificialSequence template 9 ccatctcttc ctatagtgag tccggctgca 30 10 15 DNAArtificial Sequence Description of Artificial Sequence primer 10gtgccaagct taccg 15 11 43 DNA Artificial Sequence Description ofArtificial Sequence primer 11 ctgcagaatt ctaatacgac tcactataggaagagatggc gac 43 12 19 DNA Artificial Sequence Description of CombinedDNA/RNA Molecule The n at position 8 is adenosine ribonucleotide. 12tcactatngg aagagatgg 19 13 43 DNA Artificial Sequence Description ofCombined DNA/RNA Molecule The n at position 28 is adenosineribonucleotide. 13 gggacgaatt ctaatacgac tcactatngg aagagatggc gac 43 1450 DNA Artificial Sequence Description of Artificial Sequence DNA enzymedomain 14 tcacacatct ctgaagtagc gccgccgtat gtgacgctag gggttcgcct 50 1550 DNA Artificial Sequence Description of Artificial Sequence DNA enzymedomain 15 ggggggaacg ccgtaacaag ctctgaacta gcggttgcga tatagtcgta 50 1650 DNA Artificial Sequence Description of Artificial Sequence DNA enzymedomain 16 cgggactccg tagcccattg ctttttgcag cgtcaacgaa tagcgtatta 50 1750 DNA Artificial Sequence Description of Artificial Sequence DNA enzymedomain 17 ccaccatgtc ttctcgagcc gaaccgatag ttacgtcata cctcccgtat 50 1850 DNA Artificial Sequence Description of Artificial Sequence DNA enzymedomain 18 gccagattgc tgctaccagc ggtacgaaat agtgaagtgt tcgtgactat 50 1950 DNA Artificial Sequence Description of Artificial Sequence DNA enzymedomain 19 ataggccatg ctttggctag cggcaccgta tagtgtacct gcccttatcg 50 2050 DNA Artificial Sequence Description of Artificial Sequence DNA enzymedomain 20 tctgctctcc tctattctag cagtgcagcg aaatatgtcg aatagtcggt 50 2150 DNA Artificial Sequence Description of Artificial Sequence DNA enzymedomain 21 ttgcccagca tagtcggcag acgtggtgtt agcgacacga taggcccggt 50 2250 DNA Artificial Sequence Description of Artificial Sequence DNA enzymedomain 22 ttgctagctc ggctgaactt ctgtagcgca accgaaatag tgaggcttga 50 23107 DNA Artificial Sequence Description of Combined DNA/RNA Molecule Then at position 28 is adenosine ribonucleotide. 23 gggacgaatt ctaatacgactcactatngg aagagatggc gacatctcnn nnnnnnnnnn 60 nnnnnnnnnn nnnnnnnnnnnnnnnnnngt gacggtaagc ttggcac 107 24 49 DNA Artificial SequenceDescription of Artificial Sequence DNA enzyme 24 ccgcccacct cttttacgagcctgtacgaa atagtgctct tgttagtat 49 25 48 DNA Artificial SequenceDescription of Artificial Sequence DNA enzyme 25 tctcttcagc gatgcacgcttgttttaatg ttgcacccat gttagtga 48 26 46 DNA Artificial SequenceDescription of Artificial Sequence DNA enzyme 26 tctcatcagc gattgaaccacttggtggac agacccatgt tagtga 46 27 49 DNA Artificial SequenceDescription of Artificial Sequence DNA enzyme 27 ccgcccacct cttttacgagcctgtacgaa atagtgttct tgttagtat 49 28 49 DNA Artificial SequenceDescription of Artificial Sequence DNA enzyme 28 ccgcccacct cttttacgagcctgtacgaa atagtgctct cgttagtat 49 29 48 DNA Artificial SequenceDescription of Artificial Sequence DNA enzyme 29 tctcagactt agtccatcacactctgtgca tatgcctgct tgatgtga 48 30 42 DNA Artificial SequenceDescription of Artificial Sequence DNA enzyme 30 ctctcatctg ctagcacgctcgaatagtgt cagtcgatgt ga 42 31 40 DNA Artificial Sequence Description ofArtificial Sequence DNA enzyme 31 tacagcgatt cacccttgtt taagggttacacccatgtta 40 32 40 DNA Artificial Sequence Description of ArtificialSequence DNA enzyme 32 atcagcgatt aacgcttgtt tcaatgttac acccatgtta 40 3340 DNA Artificial Sequence Description of Artificial Sequence DNA enzyme33 ttcagcgatt aacgcttatt ttagcgttac acccatgtta 40 34 40 DNA ArtificialSequence Description of Artificial Sequence DNA enzyme 34 atcagcgattcacccttgtt ttaaggttgc acccatgtta 40 35 40 DNA Artificial SequenceDescription of Artificial Sequence DNA enzyme 35 atcagcgatt cacccttgtttaagcgttac acccatgttg 40 36 40 DNA Artificial Sequence Description ofArtificial Sequence DNA enzyme 36 atcagcgatt cacccttgtt ttaaggttacacccatgtta 40 37 40 DNA Artificial Sequence Description of ArtificialSequence DNA enzyme 37 atcagcgatt aacgcttatt ttagcgttac acccatgtta 40 3840 DNA Artificial Sequence Description of Artificial Sequence DNA enzyme38 atcagcgatt aacgcttgtt ttagtgttgc acccatgtta 40 39 40 DNA ArtificialSequence Description of Artificial Sequence DNA enzyme 39 atcagcgattaacgcttatt ttagcattac acccatgtta 40 40 10 DNA Artificial SequenceDescription of Artificial Sequence substrate binding region 40gccatgcttt 10 41 10 DNA Artificial Sequence Description of ArtificialSequence substrate binding region 41 ctctatttct 10 42 12 DNA ArtificialSequence Description of Artificial Sequence substrate binding region 42tatgtgacgc ta 12 43 10 DNA Artificial Sequence Description of ArtificialSequence substrate binding region 43 tatagtcgta 10 44 11 DNA ArtificialSequence Description of Artificial Sequence substrate binding region 44atagcgtatt a 11 45 13 DNA Artificial Sequence Description of ArtificialSequence substrate binding region 45 atagttacgt cat 13 46 14 DNAArtificial Sequence Description of Artificial Sequence substrate bindingregion 46 aatagtgaag tgtt 14 47 11 DNA Artificial Sequence Descriptionof Artificial Sequence substrate binding region 47 ataggcccgg t 11 48 14DNA Artificial Sequence Description of Artificial Sequence substratebinding region 48 aatagtgagg cttg 14 49 12 RNA Human immunodeficiencyvirus type 1 49 guaacuagag au 12 50 98 DNA Artificial SequenceDescription of Combined DNA/RNA Molecule Positions 7-18 is RNA; theremainer of the sequence is DNA. 50 ggaaaaguaa cuagagaugg aagagatggcgacnnnnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn nnnnnnnnnn nnncggtaagcttggcac 98 51 99 DNA Artificial Sequence Description of CombinedDNA/RNA Molecule Positions 1-24 is RNA; the remainder of the sequence isDNA. 51 ggaaaaagua acuagagaug gaagagatgg cgacnnnnnn nnnnnnnnnnnnnnnnnnnn 60 nnnnnnnnnn nnnnnnnnnn nnnncggtaa gcttggcac 99 52 50 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 52ccaatagtgc tactgtgtat ctcaatgctg gaaacacggg ttatctcccg 50 53 50 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 53ccaaaacagt ggagcattat atctactcca caaagaccac ttttctcccg 50 54 50 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 54atccgtacta gcatgcagac agtctgtctg ctttttcatt actcactccc 50 55 49 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 55caattcatga tgaccaactc tgtcaacacg cgaactttta acactggca 49 56 50 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 56cttccacctt ccgagccgga cgaagttact ttttatcaca ctacgtattg 50 57 50 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 57ggcaagagat ggcatatatt caggtaactg tggagatacc ctgtctgcca 50 58 50 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 58ctagaccatt cacgtttacc aagctatggt aagaactaga atcacgcgta 50 59 50 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 59cgtacacgtg gaaaagctat aagtcaagtt ctcatcatgt acctgaccgc 50 60 50 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 60cagtgataca tgagtgcacc gctacgacta agtctgtaac ttattctacc 50 61 50 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 61accgaattaa actaccgaat agtgtggttt ctatgcttct tcttccctga 50 62 50 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 62caggtagata taatgcgtca ccgtgcttac actcgtttta ttagtatgtc 50 63 49 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 63ccctacaaca ccactgggcc caattagatt aacgctattt tataactcg 49 64 49 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 64ccaaacggtt ataagactga aaactcaatc aatagcccaa tcctcgccc 49 65 50 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 65cacatgtata cctaagaaat tggtcccgta gacgtcacag acttacgcca 50 66 50 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 66cacaacgaaa acaatcttcc ttggcatact ggggagaaag tctgttgtcc 50 67 50 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 67cacacgaaca tgtccattaa atggcattcc gtttttcgtt ctacatatgc 50 68 49 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 68cagaacgagg gtcttgtaag actacacctc ctcagtgaca ataatcctg 49 69 49 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 69cactacagcc tgatatatat gaagaacagg caacaagctt atgcactgg 49 70 50 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 70gggtacattt atgattctct tataaagaga atatcgtact cttttcccca 50 71 49 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 71ccaaagtaca ttccaacccc ttatacgtga aacttccagt agtttccta 49 72 50 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 72cttgaagatc ctcataagac gattaaacaa tccactggat ataatccgga 50 73 50 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 73cgaatagtgt ccatgattac accaataact gcctgcctat catgtttatg 50 74 50 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 74ccaagagagt atcggataca cttggaacat agctaactcg aactgtacca 50 75 48 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 75ccactgataa ataggtaact gtctcatatc tgccaatcat atgccgta 48 76 50 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 76cccaaattat aaacaattta acacaagcaa aaggaggttc attgctccgc 50 77 50 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 77caataaactg gtgctaaacc taataccttg tatccaagtt atcctccccc 50 78 50 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 78ccgaatgaca tccgtagtgg aaccttgctt ttgacactaa gaagctacac 50 79 50 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 79ccataacaaa taccatagta aagatctgca ttatattata tcggtccacc 50 80 50 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 80cagaacaaag atcagtagct aaacatatgg tacaaacata ccatctcgca 50 81 49 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 81cctttagtta ggctagctac aacgattttt ccctgcttgg caacgacac 49 82 50 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 82ctccctacgt tacaccagcg gtacgaattt tccacgagag gtaatccgca 50 83 36 DNAArtificial Sequence Description of Artificial Sequence DNA enzyme 83cggcacctct agttagacac tccggaattt ttcccc 36 84 49 DNA Artificial SequenceDescription of Artificial Sequence DNA enzyme 84 cggcacctct agttagacactccggaattt tagcctacca tagtccggt 49 85 47 DNA Artificial SequenceDescription of Artificial Sequence DNA enzyme 85 ccctttggtt aggctagctacaacgatttt tccctgcttg aattgta 47 86 51 DNA Artificial SequenceDescription of Artificial Sequence DNA enzyme 86 ccctttggtt aggctagctacaacgatttt tccctgcttg acctgttacg a 51 87 48 DNA Artificial SequenceDescription of Artificial Sequence DNA enzyme 87 cctttagtta ggctagctacaacgattttt ccctgcttgg aacgacac 48 88 50 DNA Artificial SequenceDescription of Artificial Sequence DNA enzyme 88 catggcttaa tcatcctcaatagaagacta caagtcgaat atgtcccccc 50 89 50 DNA Artificial SequenceDescription of Artificial Sequence DNA enzyme 89 caacagagcg agtatcaccccctgtcaata gtcgtatgaa acattgggcc 50 90 49 DNA Artificial SequenceDescription of Artificial Sequence DNA enzyme 90 taccgacaag gggaattaaaagctagctgg ttatgcaacc cttttcgca 49 91 49 DNA Artificial SequenceDescription of Artificial Sequence DNA enzyme 91 ctcgaaacag tgatattctgaacaaacggg tactacgtgt tcagccccc 49 92 50 DNA Artificial SequenceDescription of Artificial Sequence DNA enzyme 92 ccaataacgt aacccggttagataagcact tagctaagat gtttatcctg 50 93 50 DNA Artificial SequenceDescription of Artificial Sequence DNA enzyme 93 caatacaatc ggtacgaatccagaaacata acgttgtttc agaatggtcc 50 94 50 DNA Artificial SequenceDescription of Artificial Sequence DNA enzyme 94 gcaacaacaa gaaccaagttacatacacgt tcatctatac tgaaccccca 50 95 50 DNA Artificial SequenceDescription of Artificial Sequence DNA enzyme 95 cctttgagtt cctaaatgccgcacggtaag cttggcacac tttgactgta 50 96 49 DNA Artificial SequenceDescription of Artificial Sequence DNA enzyme 96 caaagatctc actttggaaatgcgaaatat gtatattcgc cctgtctgc 49 97 50 DNA Artificial SequenceDescription of Artificial Sequence DNA enzyme 97 ccacgtagaa ttatctgatttataacataa cgcaggataa ctctcgccca 50 98 48 DNA Artificial SequenceDescription of Artificial Sequence DNA enzyme 98 cacaagaaag tgtcgtctccagatatttga gtacaaggaa ctacgccc 48 99 50 DNA Artificial SequenceDescription of Artificial Sequence DNA enzyme 99 catgaagaaa taggacattctacaggctgg accgttacta tgcctgtagg 50 100 46 DNA Artificial SequenceDescription of Artificial Sequence DNA enzyme 100 cataggataa tcatggcgatgcttatgacg tgtacatcta tacctt 46 101 50 DNA Artificial SequenceDescription of Artificial Sequence DNA enzyme 101 cagatgatct tcctttaaagactacccttt aaagaaacat aaggtacccc 50

We claim:
 1. A method of selecting a catalytic DNA molecule that cleavesa substrate nucleic acid sequence at a specific site, comprising thefollowing steps: a. obtaining a population of single-stranded DNAmolecules; b. admixing nucleotide-containing substrate molecules withsaid population of single-stranded DNA molecules to form an admixture;c. maintaining said admixture for a sufficient period of time and underpredetermined reaction conditions to allow single-stranded DNA moleculesin said population to cause cleavage of said substrate sequences,thereby producing substrate cleavage products; d. separating saidpopulation of single-stranded DNA molecules from said substratesequences and substrate cleavage products; and e. isolatingsingle-stranded DNA molecules that cleave nucleotide-containingsubstrate at a specific site from said population.
 2. The method ofclaim 1, wherein said substrate comprises RNA.
 3. The method of claim 1,wherein said DNA molecules that cleave said substrate at a specific siteare tagged with an immobilizing agent.
 4. The method of claim 3, whereinsaid agent comprises biotin.
 5. The method of claim 4, wherein saidisolating step further comprises exposing said tagged DNA molecules to asolid surface having avidin linked thereto, whereby said tagged DNAmolecules become attached to said solid surface.
 6. A method of in vitroselection of catalytic DNA molecules that cleave phosphoester bonds in anucleic acid substrate, comprising the following steps: a. obtaining apopulation of single-stranded DNA molecules; b. introducing geneticvariation into said population to produce a variant population; c.selecting individuals from said variant population that meetpredetermined selection criteria; d. separating said selectedindividuals from the remainder of said variant population; and e.amplifying said selected individuals, thereby obtaining in vitroselected catalytic DNA molecules that cleave phosphoester bonds in anucleic acid substrate.
 7. A non-naturally-occurring catalytic DNAmolecule comprising a nucleotide sequence defining a conserved coreflanked by one or more recognition domains, variable regions, and spacerregions.
 8. The catalytic DNA molecule of claim 7, wherein saidnucleotide sequence defines a first variable region contiguous oradjacent to the 5′-terminus of the molecule, a first recognition domainlocated 3′-terminal to the first variable region, a first spacer regionlocated 3′-terminal to the first recognition domain, a first conservedregion located 3′-terminal to the first spacer region, a second spacerregion located 3′-terminal to the first conserved region, a secondconserved region located 3′-terminal to the second spacer region, asecond recognition domain located 3′-terminal to the second conservedregion, and a second variable region located 3′-terminal to the secondrecognition domain.
 9. The catalytic DNA molecule of claim 7, whereinsaid nucleotide sequence defines a first variable region contiguous oradjacent to the 5′-terminus of the molecule, a first recognition domainlocated 3′-terminal to the first variable region, a first spacer regionlocated 3′-terminal to the first recognition domain, a first conservedregion located 3′-terminal to the first spacer region, a second spacerregion located 3′-terminal to the first conserved region, a secondconserved region located 3′-terminal to the second spacer region, asecond recognition domain located 3′-terminal to the second conservedregion, a second variable region located 3′-terminal to the secondrecognition domain, and a third recognition domain located 3′-terminalto the second variable region.
 10. A catalytic DNA molecule havingsite-specific endonuclease activity, wherein said molecule includes oneor more hairpin loop structures.
 11. The catalytic DNA molecule of claim10, wherein said endonuclease activity is specific for a nucleotidesequence defining a cleavage site comprising single-stranded nucleicacid in a substrate nucleic acid sequence.
 12. The catalytic DNAmolecule of claim 10, wherein said molecule is single-stranded.
 13. Thecatalytic DNA molecule of claim 11, wherein said single stranded nucleicacid comprises RNA, DNA, modified RNA, modified DNA, nucleotide analogs,or composites thereof.
 14. The catalytic DNA molecule of claim 11,wherein said substrate nucleic acid comprises RNA, DNA, modified RNA,modified DNA, nucleotide analogs, or composites thereof.
 15. Thecatalytic DNA molecule of claim 11, wherein said endonuclease activitycomprises hydrolytic cleavage of a phosphoester bond at said cleavagesite.
 16. A catalytic DNA molecule having site-specific endonucleaseactivity, wherein said substrate nucleic acid sequence is attached tosaid catalytic DNA molecule.
 17. The catalytic DNA molecule of claim 16,wherein said endonuclease activity is specific for a nucleotide sequencedefining a cleavage site comprising single-stranded nucleic acid in asubstrate nucleic acid sequence.
 18. The catalytic DNA molecule of claim16, wherein said molecule is single-stranded.
 19. The catalytic DNAmolecule of claim 17, wherein said single stranded nucleic acidcomprises RNA, DNA, modified RNA, modified DNA, nucleotide analogs, orcomposites thereof.
 20. The catalytic DNA molecule of claim 17, whereinsaid substrate nucleic acid comprises RNA, DNA, modified RNA, modifiedDNA, nucleotide analogs, or composites thereof.
 21. The catalytic DNAmolecule of claim 17, wherein said endonuclease activity compriseshydrolytic cleavage of a phosphoester bond at said cleavage site.
 22. Acatalytic DNA molecule having site-specific endonuclease activity,wherein said catalytic DNA molecule comprises a nucleotide sequenceselected from the group consisting of: SEQ ID NOS 52 through
 101. 23.The catalytic DNA molecule of claim 22, wherein said endonucleaseactivity is enhanced by the presence of Mg²⁺.
 24. A catalytic DNAmolecule having site-specific endonuclease activity, wherein saidcatalytic DNA molecule has a substrate binding affinity of about 1 μM orless.
 25. The catalytic DNA molecule of claim 24, wherein saidendonuclease activity is specific for a nucleotide sequence defining acleavage site comprising single-stranded nucleic acid in a substratenucleic acid sequence.
 26. The catalytic DNA molecule of claim 24,wherein said molecule is single-stranded.
 27. The catalytic DNA moleculeof claim 25, wherein said single stranded nucleic acid comprises RNA,DNA, modified RNA, modified DNA, nucleotide analogs, or compositesthereof.
 28. The catalytic DNA molecule of claim 25, wherein saidsubstrate nucleic acid comprises RNA, DNA, modified RNA, modified DNA,nucleotide analogs, or composites thereof.
 29. The catalytic DNAmolecule of claim 25, wherein said endonuclease activity compriseshydrolytic cleavage of a phosphoester bond at said cleavage site.
 30. Acatalytic DNA molecule having site-specific endonuclease activity,wherein said catalytic DNA molecule binds substrate with a K_(D) of lessthan about 0.1 μM.
 31. The catalytic DNA molecule of claim 30, whereinsaid endonuclease activity is specific for a nucleotide sequencedefining a cleavage site comprising single-stranded nucleic acid in asubstrate nucleic acid sequence.
 32. The catalytic DNA molecule of claim30, wherein said molecule is single-stranded.
 33. The catalytic DNAmolecule of claim 31, wherein said single stranded nucleic acidcomprises RNA, DNA, modified RNA, modified DNA, nucleotide analogs, orcomposites thereof.
 34. The catalytic DNA molecule of claim 31, whereinsaid substrate nucleic acid comprises RNA, DNA, modified RNA, modifiedDNA, nucleotide analogs, or composites thereof.
 35. The catalytic DNAmolecule of claim 31, wherein said endonuclease activity compriseshydrolytic cleavage of a phosphoester bond at said cleavage site.
 36. Acatalytic DNA molecule having site-specific endonuclease activity,wherein said endonuclease activity is enhanced by the presence of adivalent cation.
 37. The catalytic DNA molecule of claim 36, whereinsaid divalent cation is selected from the group consisting of Pb²⁺,Mg²⁺, Mn²⁺, Zn²⁺, and Ca²⁺.
 38. The catalytic DNA molecule of claim 36,wherein said endonuclease activity is specific for a nucleotide sequencedefining a cleavage site comprising single-stranded nucleic acid in asubstrate nucleic acid sequence.
 39. The catalytic DNA molecule of claim36, wherein said molecule is single-stranded.
 40. The catalytic DNAmolecule of claim 38, wherein said single stranded nucleic acidcomprises RNA, DNA, modified RNA, modified DNA, nucleotide analogs, orcomposites thereof.
 41. The catalytic DNA molecule of claim 38, whereinsaid substrate nucleic acid comprises RNA, DNA, modified RNA, modifiedDNA, nucleotide analogs, or composites thereof.
 42. The catalytic DNAmolecule of claim 38, wherein said endonuclease activity compriseshydrolytic cleavage of a phosphoester bond at said cleavage site.
 43. Acatalytic DNA molecule having site-specific endonuclease activity,wherein said endonuclease activity is enhanced by the presence of amonovalent cation.
 44. The catalytic DNA molecule of claim 43, whereinsaid monovalent cation is selected from the group consisting of Na⁺ andK⁺.
 45. The catalytic DNA molecule of claim 43, wherein saidendonuclease activity is specific for a nucleotide sequence defining acleavage site comprising single-stranded nucleic acid in a substratenucleic acid sequence.
 46. The catalytic DNA molecule of claim 43,wherein said molecule is single-stranded.
 47. The catalytic DNA moleculeof claim 45, wherein said single stranded nucleic acid comprises RNA,DNA, modified RNA, modified DNA, nucleotide analogs, or compositesthereof.
 48. The catalytic DNA molecule of claim 45, wherein saidsubstrate nucleic acid comprises RNA, DNA, modified RNA, modified DNA,nucleotide analogs, or composites thereof.
 49. The catalytic DNAmolecule of claim 45, wherein said endonuclease activity compriseshydrolytic cleavage of a phosphoester bond at said cleavage site.
 50. Acatalytic DNA molecule having site-specific endonuclease activity,wherein said catalytic DNA molecule comprises a conserved core flankedby first and second substrate binding regions and wherein one or morespacer nucleotides are present between said conserved core and saidsubstrate binding region.
 51. The catalytic DNA molecule of claim 50,wherein said conserved core comprises one or more conserved regions. 52.The catalytic DNA molecule of claim 50, wherein said first substratebinding region includes a nucleotide sequence selected from the groupconsisting of: CATCTCT; GCTCT; TTGCTTTTT; TGTCTTCTC; TTGCTGCT;GCCATGCTTT  (SEQ ID NO 40); CTCTATTTCT  (SEQ ID NO 41); GTCGGCA;CATCTCTTC; and  ACTTCT.
 53. The catalytic DNA molecule of claim 50,wherein said second substrate binding region includes a nucleotidesequence selected from the group consisting of: TATGTGACGCTA  (SEQ ID NO42); TATAGTCGTA  (SEQ ID NO 43); ATAGCGTATTA  (SEQ ID NO 44);ATAGTTACGTCAT  (SEQ ID NO 45); AATAGTGAAGTGTT  (SEQ ID NO 46);TATAGTGTA; ATAGTCGGT; ATAGGCCCGGT  (SEQ ID NO 47); AATAGTGAGGCTTG  (SEQID NO 48); and ATGNTG.
 54. The catalytic DNA molecule of claim 50,further comprising a third substrate binding region, wherein said thirdregion includes a nucleotide sequence selected from the group consistingof: TGTT; TGTTA; and TGTTAG.
 55. The catalytic DNA molecule of claim 51,wherein said one or more conserved regions includes a nucleotidesequence selected from the group consisting of: CG; CGA; AGCG; AGCCG;CAGCGAT; CTTGTTT; and CTTATTT.
 56. The catalytic DNA molecule of claim51, further comprising one or more variable or spacer nucleotidesbetween said conserved regions in said conserved core.
 57. The catalyticDNA molecule of claim 54, further comprising one or more spacer regionsbetween said substrate binding regions.
 58. The catalytic DNA moleculeof claim 54, wherein said endonuclease activity is specific for anucleotide sequence defining a cleavage site comprising single-strandednucleic acid in a substrate nucleic acid sequence.
 59. The catalytic DNAmolecule of claim 54, wherein said molecule is single-stranded.
 60. Thecatalytic DNA molecule of claim 54, wherein said single stranded nucleicacid comprises RNA, DNA, modified RNA, modified DNA, nucleotide analogs,or composites thereof.
 61. The catalytic DNA molecule of claim 58,wherein said substrate nucleic acid comprises RNA, DNA, modified RNA,modified DNA, nucleotide analogs, or composites thereof.
 62. Thecatalytic DNA molecule of claim 58, wherein said endonuclease activitycomprises hydrolytic cleavage of a phosphoester bond at said cleavagesite.
 63. A composition comprising two or more populations of catalyticDNA molecules having site-specific endonuclease activity, wherein eachpopulation of catalytic DNA molecules cleaves a different nucleotidesequence in a substrate.
 64. A composition comprising two or morepopulations of catalytic DNA molecules having site-specific endonucleaseactivity, wherein each population of catalytic DNA molecules recognizesa different substrate.
 65. A method of cleaving a phosphoester bond,comprising: a. admixing a catalytic DNA molecule capable of cleaving asubstrate nucleic acid sequence at a defined cleavage site with aphosphoester bond-containing nucleic acid substrate, to form a reactionadmixture; b. maintaining said admixture under predetermined reactionconditions to allow said catalytic DNA molecule to cleave saidphosphoester bond, thereby producing a population of nucleic acidsubstrate products; c. separating said products from said catalytic DNAmolecule; and d. adding additional substrate to said catalytic DNAmolecule to form a new reaction admixture.
 66. The method of claim 65,wherein said substrate comprises RNA.
 67. A method of cleaving aphosphoester bond, comprising: a. admixing a catalytic DNA moleculecapable of cleaving a substrate nucleic acid sequence at a definedcleavage site with a phosphoester bond-containing nucleic acidsubstrate, to form a reaction admixture; and b. maintaining saidadmixture under predetermined reaction conditions to allow saidcatalytic DNA molecule to cleave said phosphoester bond, therebyproducing a population of nucleic acid substrate products, wherein saidpredetermined reaction conditions include the presence of a monovalentcation, a divalent cation, or both.
 68. The method of claim 67, whereinsaid substrate comprises RNA.